Mutants of streptococcal toxin a and methods of use

ABSTRACT

This invention is directed to mutant SPE-A toxins or fragments thereof, vaccine and pharmaceutical compositions, and methods of using the vaccine and pharmaceutical compositions. The preferred SPE-A toxin has at least one amino acid change and is substantially non-lethal compared with the wild type SPE-A toxin. The mutant SPE-A toxins can form vaccine compositions useful to protect animals against the biological activities of wild type SPE-A toxin.

BACKGROUND OF THE INVENTION

Streptococcus pyogenes, also known as β-hemolytic group A streptococci(GAS) is a pathogen of humans which can cause mild infections such aspharyngitis and impetigo.

Post infection autoimmune complications can occur, namely rheumaticfever and acute glomerulonephritis. GAS also causes severe acutediseases such as scarlet fever and streptococcal toxic shock syndrome(STSS). Severe GAS infections were a large problem in the U.S. andthroughout the world at the beginning of this century. In themid-forties, the number of cases and their severity decreased steadilyfor yet not completely understood reasons. However, more recently, aresurgence of serious diseases is caused by GAS has been seen such thatthere may be 10-20,000 cases of STSS each year in the United States. Asmany as 50 to 60% of these patients will have necrotizing fascitis andmyositis; 30 to 60% will die and as many as one-half of the survivorswill have limbs amputated. In 1986 and 1987 two reports described anoutbreak of severe GAS infections localized in the Rocky Mountain area.

These reports have been followed in the past few years by several othersdescribing a disease with analogous clinical presentation. The symptomsdescribed for this disease were very similar to those described fortoxic shock syndrome (TSS), and in 1992 a committee of scientists gaveto this clinical presentation the formal name of STSS, and set thecriteria for its diagnosis. STSS is defined by the presence of thefollowing:

-   -   1. hypotension and shock;    -   2. isolation of group A streptococci;    -   3. two or more of the following symptoms: fever 38.5° C. or        higher, scarlet fever rash, vomiting and diarrhea, liver and        renal dysfunction, adult respiratory distress syndrome, diffuse        intravascular coagulation, necrotizing fascitis and/or myositis,        bacteremia.

Streptococcal isolates from STSS patients are predominantly of M type 1and 3, with M18 and nontypable organisms making up most of the reminder.The majority of M1, 3, 18, and nontypable organisms associated with STSSmake pyrogenic exotoxin A (SPE-A, scarlet fever toxin A). In contrast,only 15% of general streptococcal isolates produce this toxin. Moreover,administration of SPE-A to a rabbit animal model and in two accidentalhuman inoculations can reproduce the symptoms of STSS.

SPE-A is a single peptide of molecular weight equal to 25,787 daltons,whose coding sequence is carried on the temperate bacteriophage T12.speA, the gene for SPE-A, has been successfully cloned and expressed inEscherichia coli.

SPE-A is a member of a large family of exotoxins produced bystreptococci and staphylococci, referred to as pyrogenic toxins basedupon their ability to induce fever and enhance host susceptibility up to100,000 fold to endotoxin.

Recently these toxins have been referred to as superantigens because oftheir ability to induce massive proliferation of T lymphocytes,regardless of their antigenic specificity, and in a fashion dependent Onthe composition of the variable part of the β chain of the T cellreceptor. These toxins also stimulate massive release of IFN-γ, IL-1,TNF-α and TNF-β. Other members of this family are streptococcalpyrogenic exotoxins type B and C, staphylococcal toxic shock syndrometoxin 1, staphylococcal enteroxtoxins A, B, Cn, D, E, G and H, andnon-group A streptococcal pyrogenic exotoxins. These toxins have similarbiochemical properties, biological activities and various degrees ofsequence similarity.

The most severe manifestations of STSS are hypotension and shock, thatlead to death. It is generally believed that leakage of fluid from theintravascular to the interstitial space is the final cause ofhypotension, supported by the observation that fluid replacement therapyis successful in preventing shock in the rabbit model of STSS describedabove. It has been hypothesized that SPE-A may act in several ways onthe host to induce this pathology.

SPE-A has been shown to block liver clearance of endotoxin of endogenousflora's origin, by compromising the activity of liver Kuppfer cells.This appears to cause a significant increase in circulating endotoxin,that through binding to lipopolysaccharide binding protein (LBP) andCD14 signaling leads to macrophage activation with subsequent release ofTNF-α and other cytokines. Support for the role of endotoxin in thedisease is given by the finding that the lethal effects of SPE-A can beat least partially neutralized by the administration to animals ofpolymyxin B or by the use of pathogen free rabbits.

Another modality of induction of shock could be the direct activity ofthe toxin on capillary endothelial cells. This hypothesis stems from thefinding that the staphylococcal pyrogenic toxin TSST-1 binds directly tohuman umbilical cord vein cells and is cytotoxic to isolated porcineaortic endothelial cells.

Another of the toxin's modality of action includes itssuperantigenicity, in which the toxin interacts with and activates up to50% of the host T lymphocytes. This massive T cell stimulation resultsin an abnormally high level of circulating cytokines TNF-β and IFN-γwhich have direct effects on macrophages to induce release of TNF-α andIL-1. These cytokines may also be induced directly from macrophages bySPE-A through MHC class II binding and signalling in the absence of Tcells. The elevated levels of TNF-α and -β cause several effectstypically found in Gram negative induced shock, among which is damage toendothelial cells and capillary leak. However, the administration toSPE-A treated rabbits of cyclosporin A, which blocks upregulation ofIL-2 and T cell proliferation, did not protect the animals from shock,suggesting that additional mechanisms may be more important in causingcapillary leak.

Thus, there is a need to localize sites on the SPE-A moleculeresponsible for different biological activities. There is a need todevelop variants of SPE-A that have changes in biological activitiessuch as toxicity and mitogenicity. There is a need to developcompositions useful in vaccines to prevent or ameliorate streptococcaltoxic shock syndrome. There is also a need to develop therapeutic agentsuseful in the treatment of streptococcal toxic shock syndrome and otherdiseases.

SUMMARY OF THE INVENTION

This invention includes mutant SPE-A toxins and fragments thereof,vaccines and pharmaceutical compositions and methods of using vaccinesand pharmaceutical compositions.

Mutant SPE-A toxins have at least one amino acid change and aresubstantially nonlethal as compared with a protein substantiallycorresponding to a wild type SPE-A toxin. For vaccine compositions,mutant toxins also stimulate a protective immune response to at leastone biological activity of a wild type SPE-A toxin. Mutant toxins forvaccine compositions are optionally further selected to have a decreasein enhancement of endotoxin shock and a decrease in T cell mitogenicitywhen compared to the wild type SPE-A. An especially preferred mutant forvaccine compositions is one that has a change at an amino acidequivalent to amino acid 20 of a wild type SPE-A toxin. Forpharmaceutical compositions, it is preferred that a mutant toxin issubstantially nonlethal while maintaining T cell mitogenicity comparableto a wild type SPE-A toxin.

The invention also includes fragments of a wild type speA toxin andmutants of speA toxins. Fragments and peptides derived from wild typeSPE-A are mutant SPE-A toxins. Fragments can include different domainsor regions of the molecule joined together. A fragment is substantiallynonlethal when compared to a wild type SPE-A toxin. For mutant toxins, afragment has at least one amino acid change compared to a wild typeSPE-A amino acid sequence. Fragments are also useful in vaccine andpharmaceutical compositions.

The invention also includes expression cassettes, vectors andtransformed cells. An expression cassette comprises a DNA sequenceencoding a mutant SPE-A toxin or fragment thereof operably linked to apromoter functional in a host cell. DNA cassettes are preferablyinserted into a vector. Vectors include plasmids or viruses. Vectors areuseful to provide template DNA to generate DNA encoding a mutant SPE-Atoxin. DNA cassettes and vectors are also useful in vaccinecompositions. Nucleic acids encoding a mutant SPE-A toxin or fragmentthereof can be delivered directly for expression in mammalian cells. Thepromoter is preferably a promoter functional in a mammalian cell.Nucleic acids delivered directly to cells can provide for expression ofthe mutant SPE-A toxin in an individual so that a protective immuneresponse can be generated to at least one biological activity of a wildtype SPE-A toxin.

Additional vaccine composition include stably transformed cells or viralvectors including an expression cassette encoding a mutant SPE-A toxinor fragment thereof. Viral vectors such as vaccinia can be used toimmunize humans to generate a protective immune response against atleast one biological activity of a wild type SPE-A toxin. Transformedcells are preferably microorganisms such as S. aureus, E. coli, orSalmonella species spp. Transformed microorganisms either include mutantSPE-A toxin or fragment thereof on their surface or are capable ofsecreting the mutant toxin. Transformed microorganisms can beadministered as live, attenuated or heat killed vaccines.

The invention also includes methods of using vaccines and pharmaceuticalcompositions. Vaccines are administered to an animal in an amounteffective to generate a protective immune response to at least onebiological activity of a wild type SPE-A toxin. Preferably, the vaccinecompositions are administered to humans and protect against thedevelopment of STSS. Pharmaceutical compositions are used in methods ofstimulating T cell proliferation. The pharmaceutical compositions areespecially useful in the treatment of cancers that are treated withinterleukins, interferons or other immunomodulators, T cell lymphomas,ovarian and uterine cancers. A pharmaceutical composition isadministered to a patient having cancer.

The mutant SPE-A toxins and/or fragments thereof and other vaccinecompositions can be useful to generate a passive immune serum. MutantSPE-A toxins or fragments thereof, DNA expression cassettes or vectors,or transformed microorganisms can be used to immunize an animal toproduce neutralizing antibodies to at least one biological activity ofwild type SPE-A. The neutralizing antibodies immunoreact with a mutantSPE-A toxin and/or fragment thereof and the wild type SPE-A toxin.Passive immune serum can be administered to an animal with symptoms of Astreptococcal infection and STSS.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Ribbon drawing of the modeled 3-dimensional structure ofstreptococcal pyrogenic exotoxin A. Domain A and B are indicated.

FIG. 2 View of SPE-A as seen from the back in reference to the standardview seen in FIG. 1. Numbered residues are those homologous to residuesin TSST-1 evaluated for reduced systemic lethality.

FIG. 3 shows the DNA sequence (SEQ ID NO:12) and predicted amino acidsequence (SEQ ID NO:13) of the cloned SPE-A toxin from T12.

FIG. 4 T cell proliferation assay. Rabbit splenocytes were incubated in96 well microliter plates in quadruplicate with SPE-A, K16N-SPE-A, andN20D-SPE-A for 72 hours. Cells were pulsed with [³H] thymidine for 18 to24 hours, harvested onto filters, and [³H] thymidine incorporation wasmeasured in a scintillation counter. Results are expressed as counts perminute (CPM) versus concentrations of toxin in μg/ml. Data presented arefrom the most representative of three independent experiments.

FIG. 5 T cell proliferation assay. Rabbit splenocytes were incubated in96 well microtiter plates in quadruplicate with SPE-A, C87S-SPE-A,C98S-SPE-A, and C90S-SPE-A for 72 hours. Cells were pulsed with [³H]thymidine for 18 to 24 hours, harvested onto filters, and [³H] thymidineincorporation was measured in a scintillation counter. Results areexpressed as counts per minute (CPM) versus concentrations of toxin inμg/ml. Data presented are from the most representative of threeindependent experiments.

FIG. 6 T cell proliferation assay. Rabbit splenocytes were incubated in96 well microtiter plates in quadruplicate with SPE-A, K157E-SPE-A, andS195A-SPE-A for 72 hours. Cells were pulsed with [³H] thymidine for 18to 24 hours, harvested onto filters, and [³H] thymidine incorporationwas measured in a scintillation counter. Results are expressed as countsper minute (CPM) versus concentrations of toxin in μg/ml. Data presentedare from the more representative of three independent experiments.

FIG. 7. Superantigenicity of wild type SPEA compared to single mutant.Rabbit spleen cells were incubated for 4 days with SPEA or mutants atthe indicated doses. Four replicate wells were used at each dose of SPEAand mutants. On day 3, 1 μCI ³H thymidine was added to each well.Superantigenicity index=³H thymidine incorporation by splenocytes in thepresence of SPEA or mutants divided by ³H thymidine incorporation in theabsence of SPEA or mutants.

FIG. 8. Superantigenicity of wild type SPEA compared to double mutants.Methods used were those described in FIG. 7.

FIG. 9. SPE A Inhibition by Immunized Rabbit Sera. Rabbit sera fromrabbits immunized with single and double mutants was used to demonstratethe ability of the sera to neutralize splenocyte mitogenicity in thepresence of SPEA.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to mutant SPE-A toxins and fragments thereof,vaccine and pharmaceutical compositions including mutant SPE A toxins orfragments thereof, methods of preparing mutant SPE-A toxins andfragments thereof, and methods of using SPE-A toxins and fragmentsthereof.

Mutant SPE-A toxins are proteins that have at least one amino acidchange and have at least one change in a biological function comparedwith a protein substantially corresponding to a wild type SPE-A toxin.Preferably, the mutant SPE-A toxin is substantially nonlethal whencompared to a wild type SPE-A toxin at the same dose. Mutant SPE-Atoxins can be generated using a variety of methods includingsite-directed mutagenesis, random mutagenesis, conventional mutagenesis,in vitro mutagenesis, spontaneous mutagenesis and chemical synthesis.Mutant SPE-A toxins are preferably selected to: 1) ensure at least onechange in an amino acid; and 2) to have a change in at least onebiological function of the molecule preferably a decrease or eliminationof systemic lethality. The mutant toxins are useful in vaccinecompositions for protection against at least one biological activity ofSPE-A toxin such as prevention or amelioration of STSS, in methods oftreating animals with symptoms of STSS, and in methods for stimulating Tcell proliferation and in the treatment of cancer. Single and doubleSPE-A mutants were tested and the antibody to the mutants inhibited cellresponses to SPEA.

A. Mutant SPE-A Toxins or Fragments Thereof, Vaccine and PharmaceuticalCompositions

The invention includes mutant SPE-A toxins that have at least one aminoacid change and that have at least one change in a biological activitycompared with proteins that substantially correspond to and have thesame biological activities as wild type SPE-A.

Wild type SPE-A toxin is encoded by a gene speA found on bacteriophageT12. The wild type SPE-A toxin has a molecular weight of 25,787 Daltonsas calculated from the deduced amino acid sequence of the matureprotein. A DNA sequence encoding a wild type SPE-A toxin and thepredicted amino acid sequence for a wild type SPE-A toxin is shown inFIG. 3. A DNA sequence encoding a wild type SPE-A toxin has been clonedin E. coli and S. aureus. Amino acid number designations in thisapplication are made by reference to the sequence of FIG. 3 withglutamine at position 31 designated as the first amino acid. The first30 amino acids represent a leader sequence not present in the matureprotein.

A structural model of a wild type SPE-A toxin is shown in FIG. 1. Thestructural model was constructed by homology modeling usingInsight/Homology program available from BioSym Corp., San Diego, Calif.The model indicates that the wild type SPE-A toxin has several distinctstructural features. These structural features include: helix 2 (aminoacids 11-15); N-terminal alpha helix 3 (amino acids 18-26); helix 4(amino acids 64-72); central-α helix 5 (amino acids 142-158); helix 6(amino acids 193-202); Domain B beta strands including strand 1 (aminoacids 30-36), strand 2 (amino acids 44-52), strand 3 (amino acids55-62), strand 4 (amino acids 75-83), strand 5 (amino acids 95-106);Domain A beta strands including strand 6 (amino acids 117-126), strand 7(amino acids 129-135), strand 8 (amino acids 169-175), strand 9 (aminoacids 180-186), and strand 10 (amino acids 213-220). In addition,cysteine residues at residues 87, 90, and 98 may be important information of putative disulfide bonds or maintaining local 3-Dconformation.

The wild type SPE-A toxin has several biological activities. Thesebiological activities include: 1) fever; 2) STSS; 3) systemic lethalitydue to development of STSS or enhancement of endotoxin shock; 4)enhancing endotoxin shock; 5) induction of capillary leak andhypotension; 6) inducing release of cytokines such as IFN γ, IL-1, TNF-αand TNF-β; 7) binding to porcine aortic endothelial cells; 8) binding toMHC class II molecules; 9) binding to T-cell receptors; and 10) T-cellmitogenicity (superantigenicity). These activities can be assayed andcharacterized by methods known to those of skill in the art.

As used herein, the definition of a wild type SPE-A toxin includesvariants of a wild type SPE-A toxin that have the same biologicalactivity of wild type SPE-A toxin. These SPE-A toxins may have adifferent amino acid or their genes may have a different nucleotidesequence from that shown in FIG. 3 but do not have different biologicalactivities. Changes in amino acid sequence are phenotypically silent.Preferably, these toxin molecules have systemic lethality and enhanceendotoxin shock to the same degree as wild type SPE-A toxin shown inFIG. 3. Preferably these toxins have at least 60-99% homology with wildtype SPE-A toxin amino acid sequence as shown in FIG. 3 as determinedusing the SS2 Alignment Algorithm as described by Altschul, S. F., Bull.Math. Bio. 48:603 (1986). Proteins that have these characteristicssubstantially correspond to a wild type SPE A.

A mutant SPE-A toxin is a toxin that has at least one change in a aminoacid compared with a protein substantially corresponding to a wild typeSPE-A toxin. The change can be an amino acid substitution, deletion, oraddition. There can be more than one change in the amino acid sequence,preferably 1 to 6 changes. It is preferred that there are more than onechange in amino acid sequence to minimize reversion of mutant SPE-Atoxin to the wild type SPE-A toxin having systemic lethality ortoxicity. For mutant SPE-A toxins useful in vaccines, it is preferredthat the change in the amino acid sequence of the toxin does not resultin a change of the toxin's ability to stimulate an antibody responsethat can neutralize wild type SPE-A toxin. For mutant SPE-A toxinsuseful in vaccines, it is especially preferred that the mutant toxinsare recognized by polyclonal neutralizing antibodies to SPE-A toxin suchas from Toxin Technologies in Boca Raton, Fla. or Dr. Schlievert(University of Minnesota, Minneapolis, Minn.) and that the proteolyticprofile is not altered compared with wild type spe-A.

The changes in the amino acid sequence can be site-specific changes atone or more selected amino acid residues of a wild type SPE-A toxin.Site-specific changes are selected by identifying residues in particulardomains of the molecule as described previously or at locations wherecysteine residues are located. Site-specific changes at a particularlocation can optionally be further selected by determining whether anamino acid at a location or within a domain is identical to or hassimilar properties to an equivalent residue in other homologousmolecules by comparison of primary sequence homology or 3-Dconformation. Homologous molecules are known to those of skill in theart. A homologous molecule is one that can be identified by comparisonof primary sequence homology using the SS2 alignment algorithm ofAltschul et al., cited supra or a homology modelling program such asInsight/Homology from BioSym, San Diego, Calif. A homologous molecule isone that displays a significant number, typically 30-99%, of identicalor conservatively changed amino acids or has a similar three dimensionalstructure, typically RMS error for conserved regions of <2 Angstroms,when compared to a wild type SPE-A toxin.

Changes in the amino acid sequence at a particular site can be randomlymade or specific changes can be selected. Once a specific site isselected it is referred to by its amino acid number designation and bythe amino acid found at that site in the wild type SPE-A as shown inFIG. 3. The amino acid number designations made in this application areby reference to the sequence in FIG. 3 with the glutamine at position 31being counted as the first amino acid. Equivalent amino acidscorresponding to those identified at a particular site in proteinssubstantially corresponding to a wild type SPE-A toxin may havedifferent amino acid numbers depending on the reference sequence or ifthey are fragments. Equivalent residues are also those found inhomologous molecules that can be identified as equivalent to amino acidsin proteins substantially corresponding to a wild type SPE-A toxineither by comparison of primary amino acid structure or by comparison toa modelled structure as shown in FIG. 1 or by comparison to a knowncrystal structure of a homologous molecule. It is intended that theinvention cover changes to equivalent amino acids at the same or similarlocations regardless of their amino acid number designation.

If a specific substitution is selected for an amino acid at a specificsite, the amino acid to be substituted at that location is selected toinclude a structural change that can affect biological activity comparedwith the amino acid at that location in the wild type SPE-A. Thesubstitution may be conservative or nonconservative. Substitutions thatresult in a structural change that can affect biological activityinclude: 1) change from one type of charge to another; 2) change fromcharge to noncharged; 3) change in cysteine residues and formation ofdisulfide bonds; 4) change from hydrophobic to hydrophilic residues orhydrophilic to hydrophobic residues; 5) change in size of the aminoacids; 6) change to a conformationally restrictive amino acid or analog;and 7) change to a non-naturally occurring amino acid or analog. Thespecific substitution selected may also depend on the location of thesite selected. For example, it is preferred that amino acids in theN-terminal alpha helix have hydroxyl groups to interact with exposedamide nitrogens or that they be negatively charged to interact with thepartial positive charge present at the N-terminus of the α helix.

Mutant toxins may also include random mutations targeted to a specificsite or sites. Once a site is selected, mutants can be generated havingeach of the other 19 amino acids substituted at that site using methodssuch as described by Aiyar et al., Biotechniques 14:366 (1993) or Ho etal. Gene 77:51-54 (1984). In vitro mutagenesis can also be utilized tosubstitute each one of the other 19 amino acids or non-naturallyoccurring amino acids or analogs at a particular location using a methodsuch as described by Anthony-Cahill et al., Trends Biochem. Sci. 14:400(1989).

Mutant toxins also include toxins that have changes at one or more sitesof the molecule not specifically selected and that have a change inamino acids that is also not specifically selected but can be any one ofthe other 19 amino acids or a non-naturally occurring amino acid.

Substitutions at a specific site can also include but are not limited tosubstitutions with non-naturally occurring amino acids such as3-hydroxyproline, 4-hydroxyproline, homocysteine, 2-aminoadipic acid,2-aminopimilic acid, ornithine, homoarginine, N-methyllysine, dimethyllysine, trimethyl lysine, 2,3-diaminopropionic acid, 2,4-diaminobutryicacid, hydroxylysine, substituted phenylalanine, norleucine, norvaline,γ-valine and halogenated tyrosines. Substitutions at a specific site canalso include the use of analogs which use non-peptide chemistryincluding but not limited to ester, ether and phosphoryl and boronlinkages.

The mutant toxins can be generated using a variety of methods. Thosemethods include site-specific mutagenesis, mutagenesis methods usingchemicals such as EMS, or sodium bisulfite or UV irradiation, byspontaneous mutation, by in vitro mutagenesis and chemical synthesis.Methods of mutagenesis can be found in Sambrook et al., A Guide toMolecular Cloning, Cold Spring Harvard, New York (1989). The especiallypreferred method for site-specific mutagenesis is using asymmetric PCRwith three primers as described by Perrin and Gilliland, 1990. NucleicAcid Res. 18:7433.

Once a mutant SPE-A toxin is generated having at least one amino acidchange compared with a protein substantially corresponding to the wildtype SPE-A toxin, the mutant SPE-A toxin is screened for nonlethality.It is preferred that mutant SPE-A toxins selected from this screeningare substantially nonlethal in rabbits when administered using aminiosmotic pump (as described in Example 2) at the same dose or agreater dose than a wild type SPE-A toxin. A mutant SPE-A toxin orfragment thereof is substantially nonlethal if when administered to arabbit at the same dose as the wild type toxin less than about 10-20% ofrabbits die. Nonlethal mutant toxins are useful in vaccine andpharmaceutical compositions. While not meant to limit the invention, itis believed that some amino acid residues or domains that affectsystemic lethality are separable from other biological activitiesespecially T cell mitogenicity.

For mutant toxins useful in vaccine composition it is further preferredthat the mutant SPE-A toxins are screened for those that can stimulatean antibody response that neutralizes wild type SPE-A toxin activity. Amethod for selecting mutant toxins that can stimulate an antibodyresponse that neutralizes wild type SPE-A toxin activity is to determinewhether the mutant toxin immunoreacts with polyclonal neutralizingantibodies to wild type SPE-A such as available from Toxin Technologies,Boca Raton, Fla. or Dr. Schlievert. Methods of determining whethermutant SPE-A toxins immunoreact with antibodies to wild type SPE-A toxininclude ELISA, Western Blot, Double Immunodiffusion Assay and the like.

Optionally, the mutant toxins can also be screened to determine if theproteolytic profile of the mutant toxin is the same as the wild typeSPE-A toxin. In some cases, it is preferred that the mutants generateddo not substantially change the overall three-dimensional conformationof the mutant toxin compared with the wild type SPE-A toxin. One way ofexamining whether there has been a change in overall conformation is tolook at immunoreactivity of antibodies to wild type SPE-A toxin and/orto examine the proteolytic profile of mutant SPE-A toxins. Theproteolytic profile can be determined using such enzymes as trypsin,chymotrypsin, papain, pepsin, subtilisin and V8 protease in methodsknown to those of skill in the art. The proteolytic profile of wild typeSPE-A with the sequence shown in FIG. 3 is known. The mutants that havea similar profile to that of wild type SPE-A are selected.

Optionally, mutant toxins can also be screened and selected to haveother changes in biological activity. As described previously, there areseveral biological activities associated with wild type SPE-A toxin.Those biological activities include: 1) fever; 2) STSS; 4) enhancementof endotoxin shock; 5) capillary leak and hypotension; 6) inducingrelease of cytokines such as IFN gamma, IL-1, TNF-α and TNF-β; 7)binding to endothelial cells; 8) binding to MHC class II molecules; 9)binding to T-cell receptors; and 10) T-cell mitogenicity(superantigenicity). These biological activities can be measured usingmethods known to those of skill in the art.

For mutant SPE-A toxins or fragments thereof useful in vaccinecompositions, it is preferred that they are substantially nontoxic andimmunoreactive with neutralizing antibodies to wild type SPE-A.Neutralizing antibodies include those that inhibit the lethality of thewild type toxin when tested in animals. Optionally, mutant SPE-A toxinscan have a change in one or more other biological activities of wildtype SPE-A toxin as described previously.

Optionally, preferred mutant toxins for vaccine compositions are furtherscreened and selected for a lack of potentiation of endotoxin shock. Thepreferred assay for examining a lack of enhancement of endotoxin shockis described in Example 4. Rabbits preferably have no demonstrablebacterial or viral infection before testing. A lack of potentiation ofor substantially no enhancement of endotoxin shock is seen when lessthan about 25% of the animals develop shock when the mutant SPE toxin iscoadministered with endotoxin as compared to wild type SPE-A activity atthe same dose. More preferably, none of the animals develop shock.

Optionally, preferred mutants for vaccine compositions also are furtherscreened and selected for a change in T cell mitogenicity. A change inT-cell mitogenicity can be detected by measuring T-cell proliferation ina standard ³H thymidine assay using rabbit lymphocytes as described inExample 4; by measuring levels of production of cytokines such as IFNgamma or TNF-β; by determining the Vβ type of T cell response or bydetermining the interaction of the molecules with MHC class IIreceptors. The preferred method for detecting a decrease in T-cellmitogenicity is to measure T-cell proliferation of rabbit lymphocytes inthe presence and absence of the mutant toxin. Responses of T cells towild type SPE-A toxin is greatly enhanced above a normal in vitroresponse to an antigen. A substantial decrease in T cell mitogenicity isseen when the mutant SPE-A toxin does not stimulate a T cellproliferative response greater than the stimulation with an antigen ornegative control. Preferably, a decrease is seen such that the T cellproliferation response to the mutant SPE-A toxin is no more thantwo-fold above background when measured using rabbit lymphocytes at thesame dose as the wild type SPE-A toxin.

Optionally, the mutant SPE-A toxins useful in vaccine compositions arefurther screened and selected for a decrease in capillary leak inendothelial cells. The preferred method is using porcine aorticendothelial cells as described by Lee t el., J. Infect. Dis. 164:711(1991).

A decrease in capillary leak in the presence of mutant SPE-A toxins canbe determined by measuring a decrease in release of a radioactivelylabelled compound or by a change in the transport of a radioactivelylabelled compound. A decrease in capillary leak is seen when the releaseor transport of a radioactively labelled compound is decreased to lessthan about two fold above background when compared with the activity ofa wild type toxin.

The especially preferred mutant SPE-A toxins useful in vaccinecompositions are not biologically active compared with proteins thathave wild type SPE-A toxin activity. By nonbiologically active, it ismeant that the mutant toxin has little or no systemic lethality, haslittle or no enhancement of endotoxin shock and little or no T cellmitogenicity. Preferably, the mutant SPE-A toxins selected for vaccinecompositions substantially lack these biological activities, i.e., theyreact like a negative control or they stimulate a reaction no more thantwo-fold above background.

Changes in other biological activities can be detected as follows.Binding to MHC class II molecules can be detected using such methods asdescribed by Jardetzky, Nature 368:711 (1994). Changes in fever can bedetected by monitoring temperatures over time after administration ofthe mutant SPE-A toxins. Changes in the levels of cytokine production inthe presence of mutant SPE-A toxins can be measured using methods suchas are commercially available and are described by current protocols inimmunology. (Ed. Coligan, Kruisbeck, Margulies, Shevach, and Stroker.National Institutes of Health, John Wiley and Sons, Inc.)

Specific examples of mutant SPE-A toxins that have at least one aminoacid change and that are substantially nontoxic are described.

The especially preferred mutants for vaccine compositions are mutantSPE-A toxins that immunoreact with polyclonal neutralizing antibodies towild type SPE-A toxin, are nontoxic, and optionally have a decrease inpotentiation of endotoxin shock and a decrease in T-cell mitogenicity.The especially preferred mutants have a change in the asparagine atamino acid 20 such as the mutant N20D that has an aspartic acidsubstituted for asparagine at residue 20 in the mature toxin (N20D). TheN20D mutant has been shown to be nontoxic, to have no enhancement ofendotoxin shock and a 5-fold decrease in T cell mitogenicity. Inaddition, changes at amino acid 98 that result in a lack of a cysteinegroup at that location also result in a mutant toxin that has a decreasein enhancement in endotoxin shock and a four-fold decrease inmitogenicity. The especially preferred mutants at this location have aserine substituted for a cysteine (C98S).

The preferred mutants for stimulation of T-cell proliferation and in thetreatment of cancer are those mutant toxins that are substantiallynonlethal. It is preferred that these mutant toxins retain T-cellmitogenicity at least at the level of wild type SPE-A toxin. Theespecially preferred mutants have an amino acid change at residue 157 ofthe wild type SPE-A such as substitution of glutamic acid for lysine atthat residue (K157E). The K157E mutant has been shown to be nonlethalbut retains mitogenicity comparable to the wild type SPE-A toxin.

Mutants can be generated to affect a functional change by changing aminoacids in a particular domain of a molecule as follows. A molecular modelof wild type SPE-A toxin is shown in FIG. 1. The especially preferreddomains include the N-terminal α helix 3 (amino acids 18-26), thecentral α helix 5 (amino acids 142-158), the Domain B beta strands(amino acids 30-36; 44-52; 55-62; 75-83; and 95-106), and the Domain Abeta strands (amino acids 117-126; 129-135; 169-175; 180-186; and213-220). Cysteine residues at positions 87, 90, and 98 may also beimportant.

While not meant to limit the invention, it is believed that thesedomains form specific 3-D conformations that are important in thebiological functions of the wild type SPE-A activity. As can be seen inFIG. 2, the N-terminal α helix and central α helix are closely situatedso that residues here may be especially important in the toxicity ofwild type SPE-A molecules. In addition, amino acids in the bordering Bstrands that are in close proximity to the central alpha helix may alsobe important in toxicity. The molecular models as shown in FIGS. 1 and 2help to identify surface residues and buried residues of the structuraldomains.

For vaccine compositions, changes are preferably made to the residues inN-terminal alpha helix 3 (residues 18-26) are screened and selected todecrease systemic lethality or enhancement of endotoxin or T cellmitogenicity or all three.

A specific example of a change in the N-terminal alpha helix 3 is achange in amino acid at residue 20. A change at this residue fromasparagine to aspartic acid results in a decrease in enhancement ofendotoxin shock, a decrease in systemic lethality, and a five-folddecrease in mitogenicity. Other changes at residue 20 are preferablythose that change the distribution of charge at the surface residues orthat change the interaction of the N-terminal α helix with the central αhelix. Substitutions at amino acid 20 with charged amino acids such asglutamic acid, lysine, arginine are likely to have the same effect.Changes made in this region are preferably those that decrease insystemic lethality due to STSS.

Preferably, changes are also made in the central α helix 5 residues142-158. Mutants in this region having at least one amino acid changeare preferably selected for a decrease systemic lethality due to STSS. Asimilar central α helix identified in other toxin molecules has beenshown to be associated with toxicity. A specific example is a change atresidue 157. Change at this residue from lysine to glutamic acid resultsin a decrease in enhancement of endotoxin shock and systemic lethalitydue to STSS.

However, T-cell mitogenicity is not affected by a change at thisresidue. These results show that toxicity and enhancement of endotoxinshock are separable activities from T cell mitogenicity. For vaccinecompositions, other mutant toxins with changes in this domain areoptionally screened and selected for a decrease in T cell mitogenicity.A change in the type of charge present at amino acid 157 indicates thata substitution of aspartic acid for the lysine is likely to have asimilar effect.

Preferably changes in domain B beta strands including residues 30-36(beta strand 1), residues 44-52 (beta strand 2), residues 55-62 (betastrand 3), residues 75-83 (beta strand 4), and residues 95-106 (betastrand 5) (domain 5) are screened and selected for nonlethality, andoptionally for a decrease in enhancement of endotoxin shock and/or Tcell mitogenicity. Multiple residues that form N-terminal barrel of betasheet in several toxins such as SEB, SEA, TSST-1 have been shown to beimportant for binding to MHC class II molecules. A decrease in MHC classII binding by mutant toxins can also be selected by using assays such asdescribed by Jardetzky et al., cited supra. Changes to these residuesthat would disrupt beta sheet conformation or change the contactresidues with MHC class II molecules, especially those on the concavesurface of the beta barrel, are selected. See FIG. 1. For vaccinecompositions, it is preferred that changes that may change localconformation do not change the immunoreactivity of the mutant toxinswith polyclonal neutralizing antibodies to the wild type SPE-A toxin.

Preferably changes to Domain A beta strands, including residues 117-126(domain beta strand 6), residues 129-135 (domain 7), residues 169-175(domain 8), residues 190-186 (domain 9), and residues 213-220 (domain10), are selected to be nonlethal, have a decrease in endotoxin shock,and/or have a decrease in T cell mitogenicity. Changes that would alterthe beta sheet conformation without changing the immunoreactivity of themutant SPE-A toxin with polyclonal neutralizing antibodies to wild typeSPE-A toxin are preferably selected.

Mutant SPE-A toxins with changes to cysteine residues or introduction ofdisulfide bonds can be selected that have a decrease in lethality, oroptionally a decrease in enhancement of endotoxin shock, and/or adecrease in T cell mitogenicity. A specific example is change at thecysteine residue 98. A change at this residue from cysteine to serineresults in a mutant toxin with a decrease in mitogenicity aboutfour-fold and a decrease in enhancement in endotoxin shock and adecrease in lethality due to STSS. Changes that eliminate the cysteinegroup at residue 98 can effect biological activity in a similar manneras a substitution with serine. Other changes that could be made atresidue 98 include substitution of the other small aliphatic residuessuch as alanine, glycine or threonine. Changes at other cysteineresidues at amino acid residues 90 and 97 result in a decrease inmitogenicity.

Advantageously, mutant SPE-A toxins useful in treatment methods can begenerated that have more than one change in the amino acid sequence. Itwould be desirable to have changes at more than one location to minimizeany chance of reversion to a molecule having toxicity or lethality. Forvaccine compositions, it is desirable that a mutant toxin with multiplechanges can still generate a protective immune response against wildtype SPE-A and/or immunoreact with neutralizing polyclonal antibodies towild type SPE-A. For pharmaceutical compositions, it is preferred thatmutants with multiple changes are substantially nonlethal whilemaintaining mitogenicity for T cells. It is especially preferable tohave about 2 to 6 changes. Examples of such mutants include those withthe N20D mutation including double mutants such as N20D/K157E,N20D/C98S, triple mutants, and the like.

Double mutants of SPEA may offer advantages over single mutants. Thiswas evaluated in three experiments detailed in Example 6. Results areprovided in FIGS. 7-9. The data indicated that the N20D/C98S mutant hadless toxicity than the single N20D mutant and the double mutantN20D/K1S7E was intermediate between the other two proteins. All threemutants were significantly less toxic than wild type SPEA. Sera fromrabbits immunized with the single and double mutants inhibitedlymphocyte proliferation in response to nonmutated SPEA toxin.Lymphocyte proliferation is associated with and necessary for fulltoxicity of the toxin.

Animals were immunized against N20D, N20D/C98S, or N20D/K157E, asdescribed in Example 7. Results are provided in Table 9. Animalsimmunized with either double mutant were completely protected from feverand enhanced susceptibility to endotoxin shock.

Triple mutants are also contemplated in this application and in oneembodiment, the SPE-A mutant N20D/C98S/D45N is tested using the methodsand assays of Examples 1-7 and the primers disclosed herein.

It may also be preferable to delete residues at specific sites such asdeletion of amino acid residue 20 asparagine and/or deletion of aminoacid 157 lysine or 98 cysteine. For vaccine compositions, mutants withdeletions would be selected that immunoreact with polyclonalneutralizing antibodies to wild type SPE-A toxin and/or can stimulate aprotective immune response against wild type SPE-A activity.

Mutant toxins of SPE-A are useful to form vaccine compositions. Thepreferred mutants for vaccine compositions have at least one amino acidchange, are nontoxic systemically, and immunoreact with polyclonalneutralizing antibodies to wild type SPE-A. The especially preferredmutants include those mutant SPE-A toxins with a change at amino acid 20such as N20D, N20D/K157E, N20D/C98S, and mutants with a deletion atresidue 20 asparagine.

Mutant toxins are combined with a physiologically acceptable carrier.Physiologically acceptable diluents include physiological salinesolutions, and buffered saline solutions at neutral pH such as phosphatebuffered saline. Other types of physiological carriers include liposomesor polymers and the like. Optionally, the mutant toxin can be combinedwith an adjuvant such as Freund's incomplete adjuvant, Freund's Completeadjuvant, alum, monophosphoryl lipid A, alum phosphate or hydroxide,QS-21 and the like. Optionally, the mutant toxins or fragments thereofcan be combined with immunomodulators such as interleukins, interferonsand the like. Many vaccine formulations are known to those of skill inthe art.

The mutant SPE-A toxin or fragment thereof is added to a vaccineformulation in an amount effective to stimulate a protective immuneresponse in an animal to at least one biological activity of wild typeSPE-A toxin. Generation of a protective immune response can be measuredby the development of antibodies, preferably antibodies that neutralizethe wild type SPE-A toxin. Neutralization of wild type SPE-A toxin canbe measured including by inhibition of lethality due to wild type SPE-Ain animals. In addition, a protective immune response can be detected bymeasuring a decrease in at least one biological activity of wild typeSPE-A toxins such as amelioration or elimination of the symptoms ofenhancement of endotoxin shock or STSS. The amounts of the mutant toxinthat can form a protective immune response are about 0.1 μg to 100 mgper kg of body weight more preferably about 1 μg to about 100 μg/kg bodyweight. About 25 μg/kg of body weight of wild type SPE-A toxin iseffective to induce protective immunity in rabbits.

The vaccine compositions are administered to animals such as rabbits,rodents, horses, and humans. The preferred animal is a human.

The mutant SPE-A toxins are also useful to form pharmaceuticalcompositions. The pharmaceutical compositions are useful in therapeuticsituations where a stimulation of T-cell proliferation may be desirable,such as in the treatment of cancer. The preferred mutant SPE-A toxinsare those that are nonlethal while maintaining T-cell mitogenicitycomparable to wild type SPE-A toxin. Preferred mutants are those thathave a change at residue 157 lysine of wild type SPE-A toxins such asK157E.

A pharmaceutical composition is formed by combining a mutant SPE-A toxinwith a physiologically acceptable carrier such as physiological saline,buffered saline solutions at neutral pH such as phosphate bufferedsaline. The mutant SPE-A toxin is combined in an amount effective tostimulate T-cell proliferation comparable to wild type SPE-A toxin atthe same dose. An enhancement in T-cell responsiveness can be measuredusing standard ³H thymidine assays with rabbit lymphocytes as well as bymeasuring T-cell populations in vivo using fluorescence activated T-cellsorters or an assay such as an ELISPOT. An effective amount can also bean amount effective to ameliorate or decrease the growth of cancercells. This can be determined by measuring the effect of the mutantSPE-A toxin on growth of cancer cells in vivo or by measuring thestimulation of cancer-specific T-cells. The range of effective amountsare 100 ng to 100 mg per kg of body weight, more preferably 1 μg to 1mg/kg body weight. About 10⁻⁶ μg of wild type SPE-A toxin can stimulateenhanced T cell responsiveness. For example, these mutant SPE-A toxinscould be used either alone or in conjunction with interleukin orinterferon therapy.

The invention also includes fragments of SPE-A toxins and fragments ofmutant SPE-A toxins. For vaccine compositions, fragments are preferablylarge enough to stimulate a protective immune response. A minimum sizefor a B cell epitope is about 4-7 amino acids and for a T cell epitopeabout 8-12 amino acids. The total size of wild type SPE-A is about 251amino acids including the leader sequence. Fragments are peptides thatare about 4 to 250 amino acids, more preferably about 10-50 amino acids.

Fragments can be a single peptide or include peptides from differentlocations joined together. Preferably, fragments include one or more ofthe domains as identified in FIG. 1 and as described previously. It isalso preferred that the fragments from mutant SPE-A toxins have at leastone change in amino acid sequence and more preferably 1-6 changes inamino acid sequence when compared to a protein substantiallycorresponding to a wild type SPE-A toxin.

Preferably, fragments are substantially nonlethal systemically.Fragments are screened and selected to have little or no toxicity inrabbits using the miniosmotic pump model at the same or greater dosagethan a protein having wild type-SPE-A toxin activity as describedpreviously. It is also preferred that the fragment is nontoxic in humanswhen given a dose comparable to that of a wild type SPE-A toxin.

For vaccine compositions, it is preferred that the fragments includeresidues from the central α helix and/or the N-terminal α helix. It isespecially preferred that the fragment include a change at amino acidresidues is equivalent to residue 20 in wild type SPE-A toxin such asN20D or a change at an amino acid residue equivalent to residue 98cysteine in a wild type SPE-A toxin.

For vaccine compositions, it is preferable that a fragment stimulate aneutralizing antibody response to a protein having wild type SPE-A toxinactivity. A fragment can be screened and selected for immunoreactivitywith polyclonal neutralizing antibodies to a wild type SPE-A toxin. Thefragments can also be used to immunize animals and the antibodies formedtested for neutralization of wild type SPE-A toxin.

For vaccine compositions, especially preferred fragments are furtherselected and screened to be nonbiologically active. By nonbiologicallyactive, it is meant that the fragment is nonlethal systemically, induceslittle or no enhancement of endotoxin shock, and induces little or no Tcell stimulation. Optionally, the fragment can be screened and selectedto have a decrease in capillary leak effect on porcine endothelialcells.

The fragments screened and selected for vaccine compositions can becombined into vaccine formulations and utilized as described previously.Optionally, fragments can be attached to carrier molecules such asbovine serum albumin, human serum albumin, keyhole limpet hemocyanin,tetanus toxoid and the like.

For pharmaceutical compositions, it is preferred that the fragmentsinclude amino acid residues in the N-terminal Domain B β strands 1-5alone or in combination with the central α helix. It is especiallypreferred if the fragments include a change at an amino acid residueequivalent to the lysine at amino acid 157 of a wild type SPE-A toxinsuch as K157E.

For pharmaceutical compositions, it is preferred that the fragments arescreened and selected for nonlethality systemically, and optionally forlittle or no enhancement of endotoxin shock as described previously. Itis preferred that the fragments retain T cell mitogenicity similar tothe wild type SPE-A toxin. Fragments of a mutant toxin SPE-A can formpharmaceutical compositions as described previously.

Fragments of mutant SPE-A toxin can be prepared using PCR, restrictionenzyme digestion and/or ligation, in vitro mutagenesis and chemicalsynthesis. For smaller fragments chemical synthesis may be desirable.

The fragments of mutant SPE-A toxins can be utilized in the samecompositions and methods as described for mutant SPE-A toxins.

B. Methods for Using Mutant SPE-A Toxins, Vaccines Compositions orPharmaceutical Compositions.

The mutant SPE-A toxins and/or fragments thereof are useful in methodsfor protecting animals against the effects of wild type SPE-A toxins,ameliorating or treating animals with STSS, inducing enhanced T-cellproliferation and responsiveness, and treating or ameliorating thesymptoms of cancer.

A method for protecting animals against at least one biological activityof wild type SPE-A toxin involves the step of administering a vaccinecomposition to an animal to establish a protective immune responseagainst at least one biological activity of SPE-A toxin. It is preferredthat the protective immune response is neutralizing and protects againstlethality or symptoms of STSS. The vaccine composition preferablyincludes a mutant SPE-A toxin or fragment thereof that has at least oneamino acid change, that immunoreacts with polyclonal neutralizingantibodies to wild type SPE-A, and is nonlethal. The especiallypreferred mutant has a change at amino acid residue 20 asparagine suchas the mutant N20D, or N20D/K157E or N20D/C98S.

The vaccine composition can be administered to an animal in a variety ofways including subcutaneously, intramuscularly, intravenously,intradermally, orally, intranasally, ocularly, intraperitoneally and thelike. The preferred route of administration is intramuscularly.

The vaccine compositions can be administered to a variety of animalsincluding rabbits, rodents, horses and humans. The preferred animal is ahuman.

The vaccine composition can be administered in a single or multipledoses until protective immunity against at least one of the biologicalactivities of wild type SPE-A is established. Protective immunity can bedetected by measuring the presence of neutralizing antibodies to thewild type SPE-A using standard methods. An effective amount isadministered to establish protective immunity without causingsubstantial toxicity.

A mutant SPE-A toxin or fragment thereof is also useful to generateneutralizing antibodies that immunoreact with the mutant SPE-A toxin andthe wild type SPE-A toxin. These antibodies could be used as a passiveimmune serum to treat or ameliorate the symptoms in those patients thathave the symptoms of STSS. A vaccine composition as described abovecould be administered to an animal such as a horse or a human until aneutralizing antibody response to wild type SPE-A is generated. Theseneutralizing antibodies can then be harvested, purified, and utilized totreat patients exhibiting symptoms of STSS. Neutralizing antibodies towild type SPE-A toxin can also be formed using wild type SPE-A. However,wild type SPE-A must be administered at a dose much lower than thatwhich induces toxicity such as 1/50 to 1/100 of the LD₅₀ of wild typeSPE-A in rabbits.

The neutralizing antibodies are administered to patients exhibitingsymptoms of STSS such as fever, hypotension, group A streptococcalinfection, myositis, fascitis, and liver damage in an amount effectiveto neutralize the effect of SPE-A toxin. The neutralizing antibodies canbe administered intravenously, intramuscularly, intradermally,subcutaneously, and the like. The preferred route is intravenously orfor localized infection, topically at the site of tissue damage withdebridement. It is also preferred that the neutralizing antibody beadministered in conjunction with antibiotic therapy. The neutralizingantibody can be administered until a decrease in shock or tissue damageis obtained in a single or multiple dose. The preferred amount ofneutralizing antibodies typically administered is about 1 mg to 1000mg/kg, more preferably about 50-200 mg/kg of body weight.

The mutant SPE-A toxins and/or fragments thereof are also useful inpharmaceutical compositions for stimulation of T-cell proliferation,especially in the treatment of cancer. It is especially preferred thatthese pharmaceutical compositions be used in the place of or inconjunction with current therapies for cancer using interleukins,interferons or tumor necrosis factors. The mutant SPE-A toxins are alsouseful in treating T cell lymphomas, and ovarian and uterine cancer.While not meant to limit the invention, it is believed that mutant SPE-Atoxins can be selectively toxic for T lymphoma cells.

The pharmaceutical compositions include a mutant SPE-A toxin and/orfragment thereof that are nonlethal, while maintaining T cellmitogenicity. The preferred mutant SPE-A toxin is one that has a changeat amino acid residue 157 lysine such as K157E.

The pharmaceutical composition is administered to a patient havingcancer by intravenous, intramuscular, intradermal, orally,intraperitoneally, and subcutaneous routes, and the like. The preferredroute is intravenous. The pharmaceutical composition can be administeredin a single dose or multiple doses. The pharmaceutical composition isadministered in an amount that is effective to stimulate enhanced T-cellproliferative response and/or to decrease the growth of the cancerwithout substantial toxicity. The preferred amount ranges from 100 ng to100 mg/kg, more preferably 1 μg to 1 mg/kg. It is especially preferredthat the mutant SPE-A pharmaceutical compositions are administered inconjunction with or in place of therapies using interferons,interleukins, or tumor necrosis factors.

C. DNA Expression Cassettes Encoding Mutant SPE-A Toxins and Methods ofPreparation of Such DNA Expression Cassettes

The invention also includes DNA sequences and expression cassettesuseful in expression of mutant SPE-A toxins and/or fragments thereof. Anexpression cassette includes a DNA sequence encoding a mutant SPE-Atoxin and/or fragment thereof with at least one amino acid change and atleast one change in biological function compared to a proteinsubstantially corresponding to a wild type SPE-A toxin operably linkedto a promoter functional in a host cell. Expression cassettes areincorporated into transformation vectors and mutant SPE-A toxins areproduced in transformed cells. The mutant toxins can then be purifiedfrom host cells or host cell supernatants. Transformed host cells arealso useful as vaccine compositions.

Mutant SPE-A toxins or fragments thereof can also be formed by screeningand selecting for spontaneous mutants in a similar manner as describedfor site specific or random mutagenesis. Mutant SPE-A toxins can begenerated using in vitro mutagenesis or semisynthetically from fragmentsproduced by any procedure. Finally, mutant SPE-A toxins can be generatedusing chemical synthesis.

DNA Sequences Encoding Mutant SPE-A Toxins

A mutant DNA sequence encoding a mutant SPE-A toxin that has at leastone change in amino acid sequence can be formed by a variety of methodsdepending on the type of change selected. A DNA sequence encoding aprotein substantially corresponding to wild type SPE-A toxin functionsas template DNA used to generate DNA sequences encoding mutant SPE-Atoxins. A DNA sequence encoding wild type SPE-A toxin is shown in FIG. 3and has been deposited in a microorganism with ATTC Accession number69830.

To make a specific change or changes at a specific location or locationsit is preferred that PCR is utilized according to method of Perrin etal., cited supra. To target a change to a particular location, internalprimers including the altered nucleotides coding for the amino acidchange are included in a mixture also including a 5′ and 3′ flankingprimers. A 5′ flanking primer is homologous to or hybridizes to a DNAregion upstream of the translation start site of the coding sequence forwild type SPE-A. Preferably, the 5′ flanking region is upstream of thespeA promoter and regulatory region. For example, a 5′ flanking primercan be homologous to or hybridize to a region about 760 bases upstreamof the translation start site as shown in FIG. 2. An example of a 5′flanking primer which includes the SPE-A promoter in upstream regulatoryregion has a sequence of: 5′GGT GGA TTC TTG AAA CAG (SEQ ID NO:1)        BamnH1 GTG-3′

A downstream flanking primer is homologous to or hybridizes to a regionof DNA downstream of the stop codon of the coding sequence for wild typeSPE-A. It is preferred that the downstream flanking primer provides fortranscriptional and translational termination signals. For example, a 3′flanking primer can hybridize or be homologous to a region 200 basepairs downstream of the stop codon for the coding sequence of SPE-A. Anexample of a 3′ flanking primer has a sequence: (SEQ ID NO:2) 5′ CCC CCCGTC GAC GAT AAA ATA GTT GCT             SalI AAG CTA CAA GCT-3′The upstream and downstream flanking primers are present in every PCRreaction to ensure that the resulting PCR product includes the speApromoter and upstream regulatory region and transcriptional andtranslation termination signals. Other upstream and downstream primerscan readily be constructed by one of skill in the art. While preferred,it is not absolutely necessary that the native speA promoter andupstream regulatory region be included in the PCR product.

Each mutation at a particular site is generated using an internal primerincluding a DNA sequence coding for a change at a particular residue.For example, amino acid substitutions at a specific site can begenerated using the following internal primers: Mutant Internal PrimerN2OD 5′ AAA AAC CTT CAA GAT ATA (SEQ ID NO:3) TAT TTT CTT -3′ C87S5′-TCC-ACA-TAA-ATA GCT GAG (SEQ ID NO:4) ATG GTA ATA-TCC-3′ C9OS 5′-CTCTGT TAT TTA TCT GAA (SEQ ID NO:5) AAT GCA GAA-3′ C98S 5′ CCC TCC GTA GATCGA TGC (SEQ ID NO:6) ACT CCT TTC TGC-3′ K157E 5′-CTT ACA GAT AAT GAGCAA (SEQ ID NO:7) CTA TAT ACT-3′ S195A 5′-CCA GGA TTT ACT CAA GCT (SEQID NO:8) AAA TAT CTT ATG-3′ K16N 5′- CAA CTT CAC AGA TCT AGT (SEQ IDNO:9) TTA GTT AAC AAC CTT-3′ (forward primer) and 5′- T TTG AAG GTT GTTAAC TAA (SEQ ID NO:10) ACT AGA TCT GTG AAG TTG-3′ (backward primer)The underlined nucleotides indicate changes in the nucleotide sequencefrom a wild type speA gene as shown in FIG. 3.

Internal primers can be designed to generate a change at a specificlocation utilizing a DNA sequence encoding wild type SPE-A toxins suchas shown in FIG. 3. Primers can be designed to encode a specific aminoacid substitution at a specific location such as shown above. Primerscan be designed to result in random substitution at a particular site asdescribed by Rennell et al., J. Mol. Biol. 22:67 (1991). Primers can bedesigned that result in a deletion of an amino acid at a particularsite. Primers can also be designed to add coding sequence for anadditional amino acid at a particular location.

Primers are preferably about 15 to 50 nucleotides long, more preferably15 to 30 nucleotides long. Primers are preferably prepared by automatedsynthesis. The 5′ and 3′ flanking primers preferably hybridize to theflanking DNA sequences encoding the coding sequence for the wild typeSPE-A toxin. These flanking primers preferably include about 10nucleotides that are 100% homologous or complementary to the flankingDNA sequences. Internal primers are not 100% complementary to DNAsequence coding for the amino acids at location because they encode achange at that location. An internal primer can have about 1 to 4mismatches from the wild type SPE-A sequence in a primer about 15 to 30nucleotides long. Both flanking primers and internal primers can alsoinclude additional nucleotides that encode for restriction sites andclamp sites, preferably near the end of the primer. Hybridizationconditions can be modified to take into account the number of mismatchespresent in the primer in accord with known principles as described bySambrook et al. Molecular Cloning-A laboratory manual, Cold SpringHarbor Laboratory Press, (1989).

More than one internal primer can be utilized if changes at more thanone site are desired. For example, to generate a mutant having a changeat amino acid 20 asparagine and a change at amino acid 157 lysineinternal primers as shown above can be utilized in two separatereactions as described in Example 5. A PCR method for generatingsite-specific changes at more than one location is described in Aiyar etal. cited supra. Another method is described in Example 5.

In one method, a DNA sequence encoding a mutant SPE-A toxin with onechange at a particular site is generated and is then used as thetemplate to generate a mutant DNA sequence with a change at a secondsite. In the first round of PCR, a first internal primer is used togenerate the mutant DNA sequence with the first change. The mutant DNAsequence with the first change is then used as the template DNA and asecond internal primer coding for a change at a different site is usedto form a DNA sequence encoding a mutant toxin with changes in aminoacid sequences at two locations. PCR methods can be utilized to generateDNA sequences with encoding amino acid sequences with about 2 to 6changes.

The preferred PCR method is as described by Perrin et al. cited supra.Briefly, the PCR reaction conditions are: PCR is performed in a 100 ulreaction mixture containing 10 mM Tris-HCl (ph=8.3), 50 mM KCl, 1.5 mMMgCl₂, 200 uM each dNTP, 2 ng template plasmid DNA, 100 pmoles flankingprimer, 5 pmoles internal primer, and 2.5 units of Ampli Taq DNApolymerase (Perkin Elmer Cetus). In the second amplification step, thecomposition of the reaction mix is as above except for equal molarity (5pmoles each) of flanking primer and megaprimer and 1 ug template. PCR isconducted for 30 cycles of denaturation at 94° C.×1 minute, annealing at37° C. or 44° C.×2 minutes and elongation at 72° C. for 3 minutes.

The PCR products are isolated and then cloned into a shuttle vector(such as PMIN 164 as constructed by the method of Murray et al, J.Immunology 152:87 (1994) and available from Dr. Schlievert, Universityof Minnesota, Mpls, Minn.). This vector is a chimera of E. coli plasmidpBR328 which carries ampicillin resistance and the staphylococcalplasmid pE194 which confers erythromycin resistance. The ligated plasmidmixtures are screened in E. coli for toxin production using polylconalneutralizing antibodies to wild type SPE-A from Toxin Technologies, BocaRaton, Fla. or from Dr. Schlievert. The mutant SPE-A toxins aresequenced by the method of Hsiao et al., Nucleic Acid Res. 19:2787(1991) to confirm the presence of the desired mutation and absence ofother mutations.

Specific DNA sequences generated in this manner include a DNA sequencethat encodes mutant N20D and has the same coding sequence as shown inFIG. 3 except that an adenine at position 939 is changed to a guanineresidue. A DNA sequence that encodes mutant C87S has the same codingsequence of FIG. 3 except that thymine at position 1,152 is changed to aadenine and thymine at position 1,154 is changed to cytosine. A DNAsequence that encodes mutant SPE-A toxin C98S has the same codingsequence as FIG. 3 except that guanine at position 1,185 is changed tocytosine and thymine at position 1,186 is changed to guanine. A DNAsequence that encodes mutant SPE-A toxin C90S includes a sequence thathas the same coding sequence as FIG. 3 except that guanine at position1,161 is changed to a cytosine. A DNA sequence that encodes mutant SPE-Atoxin K157E includes a sequence that is the same as is the codingsequence shown in FIG. 3 but is changed at position 1,351 from adenineto guanine. A DNA sequence that encodes a mutant SPE-A toxin S195Aincludes a DNA sequence that has the same coding sequence as shown inFIG. 3 except that thymine at position 1,464 is a quanine. A DNAsequence that encodes a mutant K16N SPE-A toxin includes a sequence thatis the same as that shown in FIG. 3 except that adenine at position 941is changed to cytosine.

It will be understood by those of skill in the art that due to thedegeneracy of the genetic code a number of DNA sequences can encode thesame changes in amino acids.

The invention includes DNA sequences having different nucleotidesequences but that code for the same change in amino acid sequence.

For random mutagenesis at a particular site a series of primers aredesigned that result in substitution of each of the other 19 amino acidsor a non-naturally occurring conducted in a similar manner as describedabove or by the method described by Rennell et al., cited supra. PCRproducts are subcloned and then toxin production can be monitored byimmunoreactivity with polylconal neutralizing antibodies to wild typeSPE-A. The presence of a change in amino acid sequence can be verifiedby sequencing of the DNA sequence encoding the mutant SPE-A toxin.Preferably, mutant toxins are screened and selected for nonlethality.

Other methods of mutagenesis can also be employed to generate randommutations in the DNA sequence encoding the wild type SPE-A toxin. Randommutations or random mutagenesis as used in this context means mutationsare not at a selected site and/or are not a selected change. A bacterialhost cell including a DNA sequence encoding the wild type SPE-A toxin,preferably on pMIN 164, can be mutagenized using other standard methodssuch as chemical mutagenesis, and UV irradiation. Mutants generated inthis manner can be screened for toxin production using polyclonalneutralizing antibodies to wild type SPE-A. However, further screeningis necessary to identify mutant toxins that have at least one change ina biological activity, preferably that are nonlethal. Spontaneouslyarising mutants can also be screened for at least one change in abiological activity from wild type SPE-A.

Random mutagenesis can also be conducted using in vitro mutagenesis asdescribed by Anthony-Cahill et al., Trends Biochem. Sci. 14: 400 (1989).

In addition, mutant SPE-A toxins can be formed using chemical synthesis.A method of synthesizing a protein chemically is described in Wallace,FASEB J. 7:505 (1993).

Parts of the protein can be synthesized and then joined together usingenzymes or direct chemical condensation. Using chemical synthesis wouldbe especially useful to allow one of skill in the art to insertnon-naturally occurring amino acids at desired locations. In addition,chemical synthesis would be especially useful for making fragments ofmutant SPE-A toxins.

Any of the methods described herein would be useful to form fragments ofmutant SPE-A toxins. In addition, fragments could be readily generatedusing restriction enzyme digestion and/or ligation. The pre-ferredmethod for generating fragments is through direct chemical synthesis forfragment of 20 amino acids or less or through genetic cloning for largerfragments.

DNA sequences encoding mutant toxins, whether site-specific or random,can be further screened for other changes in biological activity fromwild type SPE-A toxin. The methods for screening for a change in atleast one biological activity are described previously. Once selectedDNA sequences encoding mutant SPE-A toxins are selected for at least onechange in biological activity, they are utilized to form an expressioncassette.

Formation of an expression cassette involves combining the DNA sequencescoding for mutant SPE-A toxin with a promoter that provides forexpression of a mutant SPE-A toxin in a host cell. For those mutantSPE-A toxins produced using PCR as described herein, the native speApromoter is present and provides for expression in a host cell.

Optionally, the DNA sequence can be combined with a different promoterto provide for expression in a particular type of host cell or toenhance the level of expression in a host cell. Preferably, the promoterprovides for a level of expression of the mutant SPE-A toxin so that itcan be detected with antibodies to SPE-A.

Other promoters that can be utilized in prokaryotic cells includeP_(LAC), P_(TAC), T7, and the like.

Once the DNA sequence encoding the mutant SPE-A toxin is combined with asuitable promoter to form an expression cassette, the expressioncassette is subcloned into a suitable transformation vector. Suitabletransformation vectors include at least one selectable marker gene andpreferably are shuttle vectors that can be amplified in E. coli and grampositive microorganisms. Examples of suitable shuttle vectors includepMIN 164, and pCE 104.

Other types of vectors include viral vectors such as the baculovirusvector, SV40, poxviruses such as vaccinia, is adenovirus andcytomegalovirus. The preferred vector is a pMIN 164 vector, a shuttlevector that can be amplified in E. coli and S. aureus.

Once a transformation vector is formed carrying an expression cassettecoding for a mutant SPE-A toxin, it is introduced into a suitable hostcell that provides for expression of the mutant SPE-A toxin. Suitablehost cells are cells that provide for high level of expression of themutant toxin while minimizing the possibility of contamination withother undesirable molecules such as endotoxin and M-proteins. Suitablehost cells include mammalian cells, bacterial cells such as S. aureus,E. coli and Salmonella spp., yeast cells, and insect cells.

Transformation methods are known to those of skill in the art andinclude protoplast transformation, liposome mediated transformation,calcium phosphate precipitation and electroporation. The preferredmethod is protoplast transformation.

Preferred transformed cells carry an expression cassette encoding amutant SPE-A toxin with a change at amino acid 20 asparagine. Such atransformed cell has been deposited with the American Type CultureCollection in Rockville, Md. The characteristics of the depositedmicroorganism is that it is a S. aureus carrying pMIN 164 including aDNA sequence encoding mutant N20D operably linked to the native speApromoter and other regulatory regions. This microorganism was depositedin accordance with the Budapest treaty and given Accession number 69831.

Another microorganism has been deposited with the ATCC. Thismicroorganism is S. aureus carrying a DNA sequence encoding the wildtype SPE-A toxin operably linked to the native speA promoter andregulatory regions. This microorganism was deposited with the ATCC inaccord with the Budapest treaty and given Accession number 69830.

Transformed cells are useful to produce large amounts of mutant SPE-Atoxin that can be utilized in vaccine compositions. A transformedmicroorganism can be utilized in a live, attenuated, or heat killedvaccine. A transformed microroganism includes mutant toxin SPE-A inamounts sufficient to stimulate a protective immune response to wildtype SPE-A. Preferably, the mutant SPE-A toxin is secreted. Themicroorganism is preferably nonpathogenic to humans and includes amutant toxin with multiple amino acid changes to minimize reversion to atoxic form. The microorganism would be administered either as a live orheat killed vaccine in accordance with known principles. Preferredmicroorganisms for live vaccines are transformed cells such asSalmonella spp.

A viral vector including an expression cassette with a DNA sequenceencoding a mutant SPE-A toxin or fragment thereof operably linked to apromoter functional in a host cell can also be utilized in a vaccinecomposition as described herein. Preferably, the promoter is functionalin a mammalian cell. An example of a suitable viral vector includes poxviruses such as vaccinia virus, adenoviruses, cytomegaloviruses and thelike. Vaccinia virus vectors could be utilized to immunize humansagainst at least one biological activity of a wild type SPE-A toxin.

The invention also includes a vaccine composition comprising an nucleicacid sequence encoding a mutant SPE-A toxin or fragment thereof operablylinked to a promoter functional in a host cell. The promoter ispreferably functional in a mammalian host cell. The nucleic acidsequence can be DNA or RNA. The vaccine composition is delivered to ahost cell or individual for expression of the mutant SPE A toxin orfragment thereof within the individuals own cells. Expression of nucleicacid sequences of the mutant SPE A toxin or fragment thereof in theindividual provides for a protective immune response against the wildtype SPE A toxin. Optionally, the expression cassette can beincorporated into a vector. A nucleic acid molecule can be administeredeither directly or in a viral vector. The vaccine composition can alsooptionally include a delivery agent that provides for delivery of thevaccine intracellularly such as liposomes and the like. The vaccinecomposition can also optionally include adjuvants or otherimmunomodulatory compounds, and additional compounds that enhance theuptake of nucleic acids into cells. The vaccine composition can beadministered by a variety of routes including parenteral routes such asintravenously, intraperitoneally, or by contact with mucosal surfaces.

Conditions for large scale growth and production of mutant SPE-A toxinare known to those of skill in the art. A method for purification ofmutant SPE-A toxins from microbial sources is as follows. S. aureuscarrying the mutant or the wild type speAs in pMIN164 are grown at 37°C. with aeration to stationary phase in dialyzable beef heart medium,containing 5 μg/ml of erythromycin. Cultures are precipitated with fourvolumes of ethanol and proteins resolubilized in pyrogen free water. Thecrude preparations are subjected to successive flat bed isoelectricfocusing separations in pH gradients of 3.5 to 10 and 4 to 6. Thefractions that are positive for toxin by antibody reactivity areextensively dialyzed against pyrogen free water, and an aliquot of eachis tested for purity by SDS polyacrylamide gel electrophoresis in 15%(weight/volume) gels. Polyclonal neutralizing antibodies to SPE-A areavailable from Toxin Technologies, Boca Raton, Fla. or Dr. Schlievert.Other methods of purification including column chromatography or HPLCcan be utilized.

This invention can be better understood by way of the following exampleswhich are representative of the preferred embodiments thereof, but whichare not to be construed as limiting the scope of the invention.

EXAMPLE 1 Cloning and Expression of SPE-A Wild Type

The gene encoding wild type SPE-A toxin (speA) was cloned from E. colias described in Johnson et al., Mol. Gen. Genet. 194:52-56 (1984).Briefly, the speA gene was identified by cloning of a HindIII digest ofPhage T12 DNA in pBR322 in E. Coli RR1. Transformants were selected byidentifying those positive for toxin production using polylconalneutralizing antisera to A toxin. A nucleotide sequence for A toxin isreported in Weeks et al, Inf. Imm. 52: 144 (1986).

A DNA sequence including the speA gene was subcloned and then expressedin S. aureus. The speA carried on a E. coli plasmid was digested withrestriction enzymes HindIII and SalI. The fragments were purified andligated into HindIII-SalI sites of pMIN 164 (available as describedpreviously). The vector pMIN 164 is a chimera of the staphylococcalplasmid pE194 (carrying erythromycin resistance) and the E. coli vectorpBR328 (carrying Amp and Tet resistance). Cloning of speA into theHindIII-SalI sites of this vector disrupts Tet resistance. The promoterpresent in this plasmid immediately upstream of the cloned gene is thenative speA promoter.

Expression of the speA gene was verified by detecting the toxin in adouble immunodiffusion assay with polyclonal neutralizing antibodies toSPE-A from Toxin prepared in the inventors laboratory.

EXAMPLE Administration and Immunization of Rabbits with RecombinantlyProduced SPE-A (wt)

Administration of recombinantly produced SPE-A to animals induces STSS.Immunization of animals with recombinantly produced SPE-A reduces thedeath rate when animals are challenged with M3 or M1 streptococci andprotects animals against STSS.

Administration of SPE-A induces STSS in rabbits. A rabbit model for STSShas been established by administration of SPE-A in subcutaneouslyimplanted miniosmotic pumps. Lee et al., Infect Immun. 59:879 (1991).These pumps are designed to release a constant amount of toxin over a7-day period, thus providing continuous exposure to the toxin.Recombinantly produced SPE-A was administered to rabbits at a total doseof 200 μg/in 0.2 ml over a 7-day period. The results indicate thatanimals treated with SPE-A developed the criteria of STSS with nearlyall animals succumbing in the 7-day period (data not shown). Thesymptoms of STSS in rabbits include weight loss, diarrhea, mottled face,fever, red conjunctiva and mucosa, and clear brown urine. As expected,control non-toxin treated animals remained healthy. Two other majorobservations were made: 1) fluid replacement provided completeprotection to the animals as expected, and 2) none of the toxin treatedanimals developed necrotizing fascitis and myositis, indicating factorsother than, or in addition to, SPE-A are required for the soft tissuedamage.

Development of the clinical features of STSS correlates withadministration of SPE-A. Rabbits injected with SPE-A positivestreptococci developed STSS whereas those injected with SPE-A negativestreptococci did not show symptoms of STSS.

It is well known that SPE-A is a variable trait made by some group Astreptococci. The gene for SPE-A is encoded by bacteriophage T12, andwell-characterized streptococcal strains were established that differonly in whether or not the SPE-A phage, referred to as T12 phage, ispresent. Streptococcal strain T25₃ cured T12 is positive for productionof SPE-A, whereas T25₃ cured is SPE-A negative.

Rabbits were injected subcutaneously with SPE-A positive streptococciT25₃ cured T12 or SPE-A negative T25₃ cured into implanted Wiffle golfballs, as described by Scott et al., Infect Immunity 39:383 (1983). Theresults are shown in Table 1. The results show that animals injectedwith SPE-A positive streptococci developed the clinical features ofSTSS, and 6/8 succumbed. The two surviving animals developed antibodiesto SPE-A. In contrast, the toxin negative strain, T25₃ cured, inducedonly fever, and no deaths were observed, even at much higher bacterialcell concentrations. As in the previous animal model experiments, noevidence of soft tissue necrosis was observed. Furthermore, thestreptococci remained localized in the golf balls, suggesting thesestreptococcal strains were not highly invasive. TABLE 1 Induction ofSTSS by speA in a Wiffle ball Rabbit Model Average Highest TreatmentTemperature (° C.) Dead/Total None 39.1 0/4 T25₃ cured T12* 41.2 6/8^(‡) T25₃ cured* 40.7 0/6 T25₃ cured⁺ 41.0 0/6*Approximately 1 × 10⁸ cells⁺Approximately 1 × 10¹¹ cells^(‡)2 survivors developed antibodies to SPE-A

Immunization with recombinantly produced SPE-A decreased death rateswhen rabbits were challenged with M1 or M3 streptococci. Rabbits wereimmunized with cloned SPE-A derived from S. aureus to prevent thepossibility of immunizing the animals with contaminating streptococcalproducts, such as M protein. Control animals were not immunized againstSPE-A. The rabbits received 50 μg of recombinantly produced SPE-A inemulsified in Freund's incomplete adjuvant subcutaneously. After 9 days,rabbits were challenged subcutaneously with 25 ml of M3 (1.4×10⁹ totalCFU) or M1 (4.2×10⁹ total CFU) streptococci grown in dialyzed beef heartmedium. The M1 and M3 streptococcal isolates are clinical isolates. TheM1 isolate is designated MNST and the M3 isolate is designated MNBY.These isolates are available from Dr. Schlievert, University ofMinnesota, Mpls. Minn.

The data presented in Table 2 show the striking results of theseexperiments. TABLE 2 Protection of Rabbits from STSS with necrotizingfascitis and myositis, induced by M3 or M1 streptococci, by priorimmunization against SPE-A Number Number of Immunizing Challenge  Alive^(‡)   Animals Agent* Agent⁺ Total 20 — M3  4/20 P << 0.001^(§) 20 SPE-AM3 16/19 17 — M1  9/17 P < 0.04^(§) 15 SPE-A M1 13/15*Animals were immunized against cloned SPE-A prepared from S. aureus;ELISA titers against SPE-A were greater than 10,000.⁺Animals were challenged subcutaneously with 1.4 × 10⁹ CFU M3 or 4.2 ×10⁹ CFU M1 streptococci in a dialyzable beef heart medium.^(‡)According to the guidelines of the University of Minnesota AnimalCare Committee, the experiment which used M3 streptococci was terminatedafter 24 h, and the experiment that used M1 streptococci was terminatedafter 48 h.^(§)P values determined by Fisher's Exact Probability Test.

As indicated 16 of 19 SPE-A immunized rabbits survived challenge with M3streptococci, whereas only 4 of 20 nonimmune animals survived. Thesurviving immune animals showed clear evidence of contained soft abscessformation, upon which examination of the fluid, was filled with PMNs.

Similar results were obtained in studies of M1 streptococci, except theM1 organisms were not as virulent as the M3 organisms (Table 2). Highernumbers of M1 streptococci were used, and a reduced death rate in therabbits was seen, even in nonimmune control animals. This may reflectthe approximately 50-fold lower SPE-A production by M1 strains comparedto M3 strains.

In contrast, none of the nonimmune animals showed abscess formation, andexamination of fluid from 2/2 animals revealed no PMN infiltrate. Theseresults show that one major difference between the SPE-A immune versusnonimmune animals appears to be whether or not an inflammatory responsecould be mounted. Prior work showed that SPE-A, as well as otherpyrogenic toxin superantigens, induce macrophages to produce high levelsof TNF-α. TNF-α greatly reduces PMN chemotaxis, apparently through downregulation of chemotactic receptors. Therefore, it is believed that theresults show that antibodies in the SPE-A immunized animals(titers >10,000 by ELISA) block the release of TNF-α from macrophages byneutralizing SPE-A, thus allowing the development of a protectiveinflammatory response. In the nonimmune animals SPE-A could cause asignificant release of TNF-α which in turn prevents development of aprotective chemotactic response.

It is important to note that all of the animals that died except oneshowed extensive soft tissue damage as evidenced by their entire sidesturning purple-black and in many cases sloughing. One animal in theimmunized group died after immunization. The lack of detectableinflammation in the tissue of these animals suggest that streptococcalfactors and not components of a host immune response causes necrotizingfascitis and myositis. Other extracellular factors may also contributeto the soft tissue damage, such as SPE B and streptolysins O and S.

All of the above data make a strong case for the causative role ofpyrogenic toxin superantigens, and particularly SPE-A, when present, inthe development of STSS.

EXAMPLE 3 Site Directed Mutagenesis of a DNA Sequence Encoding SPE-A

Locations in the SPE-A molecule important for biological activity wereidentified using site directed mutagenesis. Single amino acid changeswere introduced into various regions of the molecule as described below.

The model of the three dimensional structure of SPE-A is shown inFIG. 1. This model structure was constructed by Homology using anInsight/Homology program from BioSym Corp., San Diego, Calif. Thismolecule has several domains identified as: Corresponding Domain AminoAcids Helix 2 11-15 N terminal α-helix, helix 3 18-26 Domain B - βstrands strand 1 30-36 strand 2 44-52 strand 3 55-62 strand 4 75-83strand 5  95-106 Central α-helix, helix 5 142-158 Domain A - β strandsstrand 6 117-126 strand 7 129-135 strand 8 169-175 strand 9 180-186strand 10 213-220 Helix 4 64-72 Helix 6 193-202

Amino acid number designations are made by reference to the sequence inFIG. 3.

Amino acids were selected in each of the domains and to alter thecysteine residues in the molecule. The especially preferred regions arethe N terminal α-helix (18-26); the central α-helix (142 to 158); DomainA β strands and Domain B P strands.

Target residues for mutagenesis were chosen among the conserved aminoacids throughout the pyrogenic toxin family by comparing primary aminoacid sequence and/or 3-D conformational similarities or homologies usingcomputer programs as described previously. The changes made to each ofthe amino acids were selected to change the characteristics of the aminoacid side chain of residue at the particular site. For example, at threeof the residues (87, 90 and 98) serine was substituted for cysteine soas to alter the sulphydryl groups in the molecule. At three other aminoacid residues changes were made in the charge present at that site. Forexample, a lysine was changed to a glutamic (157) acid, lysine waschanged to asparagine (16) and asparagine was changed to aspartic acid(20).

Other amino acids may affect the interaction of the toxins with MHCClass II molecules. In another molecule, the TSST-1 N terminal β barrelstrands were important for contacts with α and β chains of MHC class IImolecules. Therefore, changes in the Domain A and Domain B β strands maybe important for controlling the interaction of these molecules with MHCClass II molecules. In addition, changes in the residues can be preparedusing random mutagenesis and substitution of each of the other 19 aminoacids at a particular location, and then selecting those mutants showingan alteration in biological activity such as lethality.

The mutant SPE-A molecules were prepared using site directed mutagenesisusing polymerase chain reaction (PCR) in which the template DNA was thecloned SPE-A gene from phage T12. These primers were utilized for eachmutation generated. Generation of each mutant involved using threeprimers as follows: an upstream 5′ flanking primer, an internal primerincluding the change in DNA sequence coding for a change in an aminoacid and a downstream flanking primer. The upstream flanking primer wasincluded in every PCR reaction and is homologous to a DNA region about760 bases upstream of the translational start site and has a sequence:5′ GGT GGA TCC TTG AAA CAG GTG CA-3′ (SEQ ID NO:11)        BamH1

The resulting PCR product includes the speA promoter and possibleupstream regulatory region. The downstream flanking primer iscomplementary to a region of DNA about 270 bases downstream of the stopcodon and has a sequence: 5′ -CCC CCC GTC GAC GAT AAA ATA GTT GCT AAG(SEQ ID NO:2)                Sal I CTA CAA GCT-3′The downstream flanking primer is present in every PCR reaction andbecause of the location of the primer the PCR product contains aputative transcription termination sequence.

Each mutation is generated using an internal primer including a DNAsequence coding for a change at a particular amino acid residue. Theinternal primers used to generate each mutant are as follows: MutantInternal Primer N2OD 5′ AAA AAC CTT CAA GAT ATA (SEQ ID NO:3) TAT TTTCTT -3′ C87S 5′-TCC-ACA-TAA-ATA GCT GAG (SEQ ID NO:4) ATG GTA ATA-TCC-3′C9OS 5′-CTC TGT TAT TTA TCT GAA (SEQ ID NO:5) AAT GCA GAA-3′ C98S 5′ CCCTCC GTA GAT CGA TGC (SEQ ID NO:6) ACT CCT TTC TGC-3′ K157E 5′-CTT ACAGAT AAT GAG CAA (SEQ ID NO:7) CTA TAT ACT-3′ S195A 5′-CCA GGA TTT ACTCAA GCT (SEQ ID NO:8) AAA TAT CTT ATG-3′ K16N 5′- CAA CTT CAC AGA TCTAGT (SEQ ID NO:9) TTA GTT AAC AAC CTT-3′ (forward primer) and 5′- T TTGAAG GTT GTT AAC TAA (SEQ ID NO:10) ACT AGA TCT GTG AAG TTG-3′ (backwardprimer)The underlined residues indicate changes in coding sequence made fromDNA sequence coding will type SPE-A.

PCR was conducted as follows: Briefly, a downstream flanking primer anda forward primer spanning the site of mutation and containing thenucleotide substitutions necessary to generate an amino acid change weremixed in unequal molarity in a standard PCR reaction. The DNA productobtained was prevalent in the strand containing the mutation. Thisproduct, or megaprimer, that can be several hundred bases long, wasisolated by electrophoresis in 1% agarose gel and eluted by the use ofthe Geneclean kit, as recommended by the manufacture (Bio 101, La Jolla,Calif.).

Briefly, the PCR reaction conditions are: PCR is performed in a 100 ulreaction mixture containing 10 mM Tris-HCl (ph=8.3), 50 mM KCl, 1.5 mMMgCl₂, 200 uM each dNTP, 2 ng template plasmid DNA, 100 pmoles flankingprimer, 5 pmoles internal primer, and 2.5 units of Ampli Taq DNApolymerase (Perkin Elmer Cetus). In the second amplification step, thecomposition of the reaction mix is as above except for equal molarity (5pmoles each) of flanking primer and megaprimer and 1 ug template. PCR isconducted for 30 cycles of denaturation at 94° C.×1 minute, annealing at37° C. or 44° C.×2 minutes and elongation at 72° C. for 3 minutes.Hybridization conditions can be varied in accord with known principlesdepending on the primer size, mismatches, and GC content.

A plasmid containing the speA cloned gene and flanking sequences wasused as a template. In the second step, the megaprimer and an upstreamflanking primer were combined in the reaction mixture in equal molarityto generate the full is length mutant speA.

The mutant speAs were digested with appropriate restriction enzymes andcloned into the shuttle vector pMIN 164. This vector is a chimera of theE. coli plasmid pBR328, which carries an ampicillin resistance gene, andthe staphylococcal plasmid pE194, which confers erythromycin resistance.The ligated plasmid mixtures were transformed, selected for, andscreened in E. coli. Clones positive for toxin production, as judged bydouble immunodiffusion assays, were sequenced by the method of Hsiaocited supra to confirm the presence of the desired mutation and theabsence of other mutations. Plasmids were then transformed in S. aureusstrain RN 4220 (available from Richard Novick, Skirball Institute, NewYork, N.Y.) for expression and production of mutant toxins.

S. aureus carrying the mutant or the wild type speAs in pMIN164 weregrown at 37° C. with aeration to stationary phase in dialyzable beefheart medium, containing 5 μg/ml of erythromycin. Cultures wereprecipitated with four volumes of ethanol and proteins resolubilized inpyrogen free water. The crude preparations were subjected to successiveflat bed isoelectric focusing separations in pH gradients of 3.5 to 10and 4 to 6. The fractions that were positive for toxin by antibodyreactivity were extensively dialyzed against pyrogen free water, and analiquot of each was tested for purity by SDS polyacrylamide gelelectrophoresis in 15% (weight/volume) gels (data not shown). Allmutants prepared were as resistant as the native toxin to treatment for60 minutes with trypsin (2 μg/μg SPE-A), and this together with theconserved reactivity to polyclonal antibodies raised against nativeSPE-A indicates that the mutations introduced do not cause grossstructural changes of the toxin. Using these methods, 7 mutants havingsingle amino acid substitutions in the amino acid sequence of SPE-A weregenerated.

EXAMPLE 4 Biological Activity Profile of Mutant SPE-A

Biological activities of the mutant toxins were evaluated and comparedto those of the wild type SPE-A. The mutant toxins were tested for theability to stimulate proliferation of T lymphocytes (superantigenicity),to enhance host susceptibility to endotoxin shock and for development oftoxic shock syndrome and lethality.

The ability to stimulate proliferation of T lymphocytes was measured as[³H] thymidine incorporation into cellular DNA of rabbit splenocytes. Astandard 4-day mitogenicity assay was performed in 96 well microtiterplates. Each well contained 2×10⁵ rabbit splenocytes resuspended in 200μl RPMI 1640 (Gibco, Grand Island. NY) supplemented with 25 mM HEPES,2.0 mM L-glutamine, 100 U penicillin, 100 μg/ml streptomycin and 2% heatinactivated FCS. 20 μl samples of exotoxins were added in quadruplicateamounts in final amounts: 1 μg to 10⁻⁵ μg/well. The background cellularproliferation was determined in quadruplicate wells by adding 20 μl RPMIto the splenocytes. After 3 days of incubation in a humidified chamberat 37° C. and 7% CO₂, 1.0 μCi (20 μl volume of 5-[methyl-3H]-thymidine(46 Ci/mmole, Amersham, Arlington Heights, Ill.) was added to each welland incubated for 18 hours. Cellular DNA was collected on glass fiberfilters and the [methyl-³H] thymidine incorporation was quantified byliquid scintillation counting. Three separate assays using threedifferent rabbit donors were performed. Exoprotein concentrations weretested in quadruplicate in each of three assays. Results are presentedas CPM.

The ability to enhance host susceptibility to endotoxin shock was testedin American Dutch Belted rabbits. Animals weighing between 1 and 2 kgwere injected in the marginal ear vein with 5 μg/kg body weight of SPE-A(equal to 1/50 LD₅₀) and challenged 4 hours later by IV injection of 1or 10 μg/kg body weight of endotoxin (about 1/100 LD₅₀) from Salmonellatyphimurium. Control rabbits received injections with PBS. The animalswere monitored after 48 hours for death.

Lethality was also measured using miniosmotic pumps implantedsubcutaneously in American Dutch Belted rabbits and containing 200 μg oftoxin. Individual proteins (200 μg) were injected in 0.2 ml PBS intominiosmotic pumps (Alzet, AlzaCo, Palo Alto, Calif.). The pump isdesigned to deliver a constant amount of toxin over a 7-day period.Rabbits were monitored 3 times daily for signs of toxic shock syndromesuch as diarrhea, erythema of conjunctivae and ears, shock and death forup to 8 days.

The results of the T cell mitogenicity studies are shown in FIGS. 4, 5and 6. The results show that the mutant N20D had a five-fold decrease insuperantigenicity or T cell mitogenicity activity. Mutants C87S and C98Salso had a 4-fold decrease in mitogenicity for T cells. Thus, several ofthe mutations affected biological activity of superantigenicity or Tcell mitogenicity.

The results of enhancement of endotoxin shock and lethality are shown inTables 3, 4, and 5 shown below. TABLE 3 Mutants SPE-A-K16N andSPE-A-N20D assayed for ability to cause endotoxin enhancement orlethality when administered in subcutaneous miniosmotic pumps. Resultsare expressed as ratio of deaths over total rabbits tested Protein SPE-AK16N N20D Endotoxin enhancement 3/3 6/7 0/3 1 μg/kg endotoxin) Lethalityin 3/4 ND 0/4 miniosmotic pumps

TABLE 4 Mutants SPE-A-C87S, SPE-A-C90S, and SPE-A-C98S tested forability to induce endotoxin enhancement or lethality when administeredin subcutaneous miniosmotic pumps. Results are expressed as ratio ofdeaths over total number of treated rabbits. Protein SPE-A C87S C98SC90S Endotoxin enhancement 2/3 1/3 0/3 ND 1 μg/kg body weight Endotoxinenhancement 2/3 3/3 1/3 ND 10 μg/kg body weight Lethality in 3/4 ND ND3/3 miniosmotic pumps

TABLE 5 Mutants SPE-A-K157E and SPE-A-S195A tested for ability to inducelethality when administered in subcutaneous miniosmotic pumps. Resultsare expressed as ratio of deaths over total number of treated rabbitsProtein SPE-A K157E S195A Lethality in 6/8 0/4 3/3 miniosmotic pumps

The results show that animals treated with the mutant N20D did notdevelop STSS when tested using either model of STSS. The mutation inN20D is located in an organized α-helix bordering the deep groove on theback of the toxin (FIG. 1). This residue is important both insuperantigenicity and lethality functions of the molecule.

Mutations that eliminated sulphydryl groups and, therefore, thatinterfere with possible disulfide linkages, have varied effects on thebiological activities of SPE-A, depending on which cysteine residue wasmutated. The C90S mutant remained completely lethal (Table 4), and Tcell stimulatory activity was not significantly decreased is (FIG. 5 a).In contrast, C87S and C98S mutations reduced approximately four fold thetoxin's mitogenicity (FIG. 5 b).

However, ability to cause endotoxin shock was affected differently bythe two mutations, with C98S being only weakly toxic, but C87S beingstrongly toxic (Table 4). An explanation for these results is based uponthe relative positions of the three cysteine residues in the primarysequence and in the 3-dimensional structure (FIG. 1). The lack of thesulfhydryl group of C98 may preclude formation of a putative disulfidebridge seen in staphylococcal enterotoxins, and therefore, theconformation of the loop would be lost. This would have detrimentaleffects for the activity if amino acids in this loop are responsible forcontact with host cellular receptors or have some other function inbiological activity of the molecule. In the case of C87S mutation, theputative disulfide bond could still be created between C90 and C98,preserving most of the conformation and, therefore, the activity.

Mutant K157E, located within the long central α-helix, retained completesuperantigenicity (FIG. 6 b), but was nonlethal when administered inminiosmotic pumps to rabbits (Table 6).

Residue S195A, which is part of α-5 helix, may not be important for thebiological activities tested, since its mutation does not affectactivities tested thus far. This residue may not be exposed to theenvironment or may not contribute to binding.

These results show that lethality and superantigenicity can be affectedby mutations at several sites. Lethality can be affected by mutations inresidues in the N terminal α-helix (N20D) and in the central α-helix(K157E). Mitogenicity can be affected by mutations in the N terminala-helix and changes to sulfhydryl groups.

These results also show that mitogenicity and lethality are separableactivities as mutants were generated that affect lethality withoutaffecting superantigenicity (K157E) and that affected mitogenicitywithout affecting lethality (C87S).

EXAMPLE 5 Preparation of Double or Triple Mutants of SPE-A Using PCR

There are a number of methods that can be used to generate double ortriple mutant SPE-A toxins or fragments thereof.

Mutant SPE-A toxins with two or more changes in amino acid sequences canbe prepared using PCR as described previously. In a first PCR reaction,an first internal primer coding for the first change at a selected siteis combined with 5′ and 3′ flanking primers to form a first PCR product.The first PCR product is a DNA sequence coding for a mutant SPE-A toxinhaving one change in amino acid sequence. This first PCR product thenserves as the template DNA to generate a second PCR product with twochanges in amino acid sequence compared with a protein having wild typeSPE-A activity. The first PCR product is the template DNA combined witha second internal primer coding for a change in amino acid at a secondsite. The second internal primer is also combined with the 5′ and 3′flanking primers to form a second PCR product. The second PCR product isa DNA sequence encoding a mutant SPE-A toxin with changes at two sitesin the amino acid sequence. This second PCR product can then be used asa template in a third reaction to form a product DNA sequence encoding amutant SPE-A toxin with changes at three sites in the amino acidsequence. This method can be utilized to generate DNA sequences encodingmutant toxins having more than one change in the amino acid sequence.

An alternative method to prepare DNA sequences encoding more than onechange is to prepare fragments of DNA sequence encoding the change orchanges in amino acid sequence by automated synthesis. The fragments canthen be subcloned into the wild type SPE-A coding sequence using severalunique restriction sites. Restriction sites are known to those of skillof the art and can be readily determined from the DNA sequence of a wildtype SPE-A toxin. The cloning can be done in a single step with a threefragment ligation method as described by Revi et al. Nucleic Acid Res.16: 1030 (1988).

EXAMPLE 6 Toxicity Studies Related to Single and Double Mutants

Wild type SPEA, SPEA N20D, SPEA K157E, SPEA N20/C98S, and SPEAN20D/K157E were evaluated for superantigenicity based on their capacityto stimulate rabbit splenocyte proliferation (see FIGS. 7 and 8).

Double mutants SPEA (N20D/C98S, N20D/K157E) were prepared by PCRmutagenesis using the techniques described above. The mutant SPEA gene,speA N20D, served as template DNA for introduction of the secondmutation. The double mutant genes were sequenced as described above toinsure that only the indicated changes were present. Only the desiredchanges were present.

Rabbit spleen cells were cultured in the presence of SPEA and SPEAmutants in vitro for 3 days and then an additional day after addition of1 μCi/well of ³H thymidine.

Incorporation of ³H thymidine into lymphocyte DNA was used as themeasure of T cell proliferation. A superantigenicity index wascalculated as average counts/min ³H thymidine incorporation instimulated cells divided by average counts/min in cells cultured withoutadded SPEA or mutants.

Wild type SPEA was significantly superantigenic at doses from 1 to 0.001μg/well (FIG. 7). SPEA K157E was significantly mitogenic at doses of0.01 and 0.001 μg/well (FIG. 7). The three other SPEA mutants (SPEAN20D, SPEA N20D/C98S, SPEA N20D/K157E) were significantly lesssuperantigenic (FIG. 8) than wild type SPEA at doses of 1 to 0.001 μg(p<0.001). Interestingly, SPEA N20D was significantly moresuperantigenic (FIG. 8) than SPEA N20D/C98S at doses of 1 and 0.1 μg(p<0.0005, p<0.001, respectively). Furthermore, SPEA N20D was moremitogenic than SPEA N20D/K157E at the 1 μg/well dose (p<0.01). Thus, thedata indicated the N20D/C98S mutant had less toxicity than the singleN20D mutant, and the double mutant N20D/K157E was intermediate betweenthe other two proteins. All three mutants were significantly less toxicthan wild type SPEA.

In a second experiment rabbits (3/group) were challenged iv with 10μg/kg SPEA or mutants and then endotoxin 5 μg/kg) 4 hours later. Animalswere monitored for 48 hours for enhanced lethality due to administrationof SPE and endotoxin. This assay is the most sensitive in vivo measureof SPEA lethal activity. As indicated in Table 6, 0/3 animals challengedwith wild type SPEA and endotoxin survived. In contrast all but oneanimal challenged with SPEA N20D survived, and all animals challengedwith SPEA N20D/C98S or SPEA N20D/K157E survived. TABLE 6 Capacity ofSPEA (10 μg/kg) or mutants (10 μg/kg) to enhance rabbit susceptibilityto the lethal effects of endotoxin (5 μg/kg) SPEA or Mutant NumberDead/Total Wild type SPEA 3/3 SPEA N20D 1/3 SPEA N20D/C98S 0/3 SPEAN20D/K157E 0/3Note:SPEA or mutants were administered iv at 0 hour and endotoxin iv at 4hours. Animals were monitored for 48 hours for lethality.

In a third experiment rabbits were immunized with SPEA N20D, SPEAN20D/C98S, OR SPEA N20D/K157E, and then challenged with wild type SPEA(10 μg/kg) and endotoxin (5 μg/kg or 25 μg/kg) as in the precedingexperiment. Control animals were not immunized but were challenged withwild type SPEA plus endotoxin. Rabbits were immunized every other weekfor two injections, with mutant proteins (50 μg/injection) emulsified inincomplete adjuvant (Freund's, Sigma Chemical Co., St. Louis, Mo.) andthen rested one week prior to challenge with wild type toxin. Thecombination of wild type SPEA and endotoxin represent 20 LD₅₀ forchallenge with 10 μg/kg SPEA and 5 μg/kg endotoxin, and 100 LD₅₀ forchallenge with 10 μg/kg SPEA and 25 μg/kg endotoxin.

As indicated in Table 7, all animals challenged with 100 LD₅₀ of SPEAand endotoxin succumbed. Similarly, all animals immunized with SPEA N20Dor N20D/K157E succumbed when challenged with 20 LD₅₀ of SPEA andendotoxin. In contrast, animals immunized with the double mutantN20D/C98S survived. Animals immunized with the double mutant N20D/K157Esuccumbed earlier than other animals. The data above indicates thatdouble mutants and in particular SPEA N20D/C98S shows effectiveness as atoxoid vaccine in test animals. TABLE 7 Ability of SPEA mutants toimmunize rabbits against the capacity of wild type SPEA to enhancesusceptibility to lethal endotoxin shock. Immunizing Challenge dose ofSPEA and Number Agent Endotoxin Dead/Total None 10 μg/kg SPEA, 25 μg/kgendotoxin 3/3 SPEA N20D 10 μg/kg SPEA, 25 μg/kg endotoxin 2/2 SPEAN20D/C98S 10 μg/kg SPEA, 25 μg/kg endotoxin 2/2 SPEA N20D/K157E 10 μg/kgSPEA, 25 μg/kg endotoxin 2/2 None 10 μg/kg SPEA, 5 μg/kg endotoxin 3/3SPEA N20D 10 μg/kg SPEA, 5 μg/kg endotoxin 2/2 SPEA N20D/C98S 10 μg/kgSPEA, 5 μg/kg endotoxin 0/3 SPEA N20D/K157E 10 μg/kg SPEA, 5 μg/kgendotoxin 3/3Note:Some animals escaped during this experiment. These animals were notincluded in the above data.

EXAMPLE 7 SPE A Inhibition by Antibodies to SPE-A Mutants and SPE-AMutant Immunization

One ml of blood was drawn from the marginal ear vein from each of therabbits immunized with N20D, N20D/C98S, and N20D/K157E SPEA andnonimmunized controls. Animals were bled 6 days after the lastimmunization (one day before animals were used in the experiment inTable 6). After the blood clotted, sera were separated by centrifugation(13,000×g, 10 min). Sera from each group were pooled and treated with33⅓% (final concentration) of ammonium sulfate for 1 hr at roomtemperature to precipitate immunoglobulins. Precipitated immunoglobulinswere collected by centrifugation (13,00×g, 10 min), resolubilized to theoriginal volume in phosphate-buffered saline (0.005M NaPO₄pH7.0, 0.15MNaCl), and dialyzed for 24 hr against 1 liter of 0.15M NaCl at 4° C. Thedialysates were filter sterilized (0.45 μm pore size) and used instudies to neutralize rabbit splenocyte mitogenicity (superantigenicity)of 0.01 μg SPEA (FIG. 9). Serum from one rabbit immunized with sublethaldoses of wild type SPEA was fractionated comparably and used as thepositive control. Twenty microliters of the immunoglobulin fractions(Igs) from each group of sera were diluted 1/5 and 1/50 with completeRPMI 1640 mammalian cell culture media (dilution with respect to theoriginal serum volume) and added to each of 4 wells containing wild typeSPEA and 2×10⁵ rabbit splenocytes in our standard mitogenicity assay.Igs and wild type toxin were both added to lymphocytes at time 0. Theresults are shown in FIG. 9.

The 1/5 diluted Igs, whether from immunized animals or nonimmunecontrols were inhibitory to splenocyte proliferation, probably becauseof residual ammonium sulfate in the Igs. However, Igs from the SPEAimmune animals and Igs from N20D, N20D/C98S, and N20D/K157E immuneanimals were more inhibitory than Igs from nonimmune controls (p=0.006for SPEA versus nonimmune, [=0.035 for N20D versus nonimmune, p=0.0002for N20D/C98S versus nonimmune, and p=0.0001 for N20D/K157E versusnonimmune by use of Student's t test analysis of normally distributedunpaired data), indicating specific inhibition of mitogenicity.

When Igs were added at the 1/50 dilution, the double mutant N20D/C98Scaused significant inhibition of splenocyte proliferation compared tononimmune controls (p=0.046). At this Ig concentration none of thefractions caused nonspecific suppression of lymphocyte mitogenicity.

These data suggest that the double mutant N20D/C98S was better able toimmunize animals against mitogenicity of the wild type SPEA than thesingle mutant N20D or the other double mutant N20D/K157E. However, thedouble mutant N20D/K157E was a better immunogen than the single mutantN20D. Without being bound by the following, it is possible the twochanges in the N20D/C98S mutant interfere with host cell receptor sitesrequired for lethality, T cell receptor interaction, and possiblyindirectly, class II MHC interaction on antigen presenting cells. Sinceclass II MHC interaction depends on amino acid residues in the P barreldomain (domain B) in the standard view of the toxin, we propose alsothat a change in this region (such as D45N) may improve theimmunogenicity of N20D/C98S even more. The basis for this hypothesis isthat wild type toxin (and possibly mutants lacking changes in the classII MHC interaction domain) bind directly to class II MHC moleculeswithout the requirement for normal processing by antigen presentingcells. Mutants that contain amino acid changes that interfere with thisdirect class II MHC interaction may be more immunogenic since themutants may be more easily internalized and processed. Thus, the triplemutant N20D/C98S/D45N will be evaluated using the methods used toevaluate the other mutants.

Sera obtained from the nonimmune controls and each group of N20D,N20D/C98S, or N20D/K157E immunized rabbits were tested directly forELISA titer against wild type SPEA (L. Hudson and F. C. Hay, PracticalImmunology 2nd Ed, 1980, Blackwell Scientific Publications, Boston p237-239.) Serum from each animal was evaluated separately. The antibodytiters obtained were averaged and are shown in Table 8. Nonimmunecontrol animals as expected had very low titers of antibodies againstSPEA. In contrast all animals immunized against the mutants hadsignificant antibody titers. The animals immunized with the doublemutant N20D/K157E had the highest average titer with the other twomutants being comparable. However, the range of titers for the N20Dimmunized animals was much greater (20, 40, 160, 640, 640 for each ofthe 6 animals) than either of the double mutants. The data suggest thedouble mutants gave more consistent immunization. TABLE 8 ELISA antibodytiters of animals immunized against N20D, N20D/C98S, N20D/K157E SPEA andnonimmune controls Immunizing Agent Average Antibody Titer^(a) Range^(b)None 10 <10-20   N20D SPEA 250  20-640 N20D/C98S SPEA 80 80 N20D/K157ESPEA 425 320-640^(a)6 animals/group^(b)The lowest titer detectable was 10. Titer is the reciprocal of thelast dilution that gave a positive result.

In a final experiment animals (3/group) were immunized against N20D,N20D/C98S, or N20D/K157E (50 μg/injection iv) by administering mutantprotein every other day for 5 injections and then resting the animalsfor one day. Animals were then evaluated for immunity against theability of wild type SPEA to cause fever [20 times the minimum pyrogenicdose (MPD) 4 hours after injection/kg body weight (20 MPD-4)]. SPEA isone of the most potent pyrogens known with one MPD-4 in rabbits of 0.15μg/kg. At the 4 hr timepoint animals were injected with endotoxin (25μg/kg) to evaluate immunity to the enhanced susceptibility to endotoxinshock. The results are shown in Table 9.

The nonimmune animals and those immunized with N20D SPEA showed bothsignificant fever responses (0.8° C. for both groups) and enhancedsusceptibility to endotoxin (⅔ succumbed in 48 hr in both groups). Incontrast animals immunized with either double mutant were completelyprotected from fever and the enhancement phenomenon.

Collectively, all of the above data suggest both double mutants arebetter able to immunize animals against the toxic effects of SPEA thanthe single mutant. None of the mutants themselves were toxic to theanimals. The double mutant N20D/C98S was a better immunogen thanN20D/K157E, but both were effective. TABLE 9 Ability of SPEA mutantsN20D, N20D/C98S, and N20D/K157E to immunize rabbits against SPEApyrogenicity and lethal challenge by SPEA and endotoxin. Fever ResponseImmunizing Agent Change ° C. at 4 hr Number Dead/Total None 0.8 2/3 N20DSPEA 0.8 2/3 N20D/C98S SPEA 0.0 0/3 N20D/K157E SPEA 0.1 0/3

Although the invention has been described in the context of particularembodiments, it is intended that the scope of coverage of the patent notbe limited to those particular embodiments, but is determined byreference to the following claims.

1. A mutant SPE-A toxin or fragment thereof, the mutant SPE-A toxincomprising one to six amino acid substitutions and being substantiallynonlethal compared with a protein substantially corresponding to wildtype SPE-A toxin; wherein at least one of the substituted amino acids ispositioned in N-terminal alpha helix 3, in domain B beta strand 1, indomain B beta strand 2, in domain B beta strand 3, in domain A betastrand 6, in domain A beta strand 8, in domain A beta strand 9, indomain A beta strand 10, or is a cysteine.
 2. The mutant SPE-A toxin ofclaim 1, wherein the mutant SPE-A toxin comprises one to six amino acidsubstitutions; and wherein at least one of the substituted amino acidsis asparagine-20, lysine-157, or cysteine-98.
 3. The mutant SPE-A toxinof claim 2, wherein the at least one amino acid substitution comprisesthe substitution of asparagine-20 to aspartic acid, glutamic acid,lysine or arginine; the substitution of cysteine 98 to serine, alanine,glycine, or threonine; or the substitution of lysine-157 to glutamicacid or aspartic acid.
 4. The mutant SPE-A toxin of claim 3, wherein theat least one amino acid substitution comprises asparagine-20 to asparticacid, cysteine 98 to serine, or lysine-157 to glutamic acid.
 5. Themutant SPE-A toxin of claim 2, wherein the at least one amino acidsubstitution comprises substitution of asparagine-20.
 6. The mutantSPE-A toxin of claim 5, wherein the substitution is asparagine-20 toaspartic acid.
 7. The mutant SPE-A toxin of claim 5, further comprisingsubstitution of cysteine-98, or lysine-157.
 8. The mutant SPE-A toxin ofclaim 7, wherein the substitution is cysteine 98 to serine, orlysine-157 to glutamic acid.
 9. The mutant SPE-A toxin of claim 1,wherein the mutant has at least one of the following characteristics:the mutant has a decrease in mitogenicity for T-cells, the mutant doesnot substantially enhance endotoxin shock, the mutant is not lethal, orthe mutant is nonlethal but retains mitogenicity comparable to that ofthe wild type SPE-A toxin.
 10. A vaccine for protecting animals againstat least one biological activity of wild-type SPE-A comprising: aneffective amount of at least one mutant SPE-A toxin according toclaim
 1. 11. A pharmaceutical composition comprising: a mutant SPE-Aaccording to claim 1 in admixture with a physiologically acceptablecarrier.
 12. A DNA sequence encoding a mutant SPE-A toxin according toclaim
 1. 13. A stably transformed host cell comprising a DNA sequenceaccording to claim
 12. 14. A method for protecting an animal against atleast one biological activity of a wild type SPE-A comprising:administering a vaccine according to claim 10 to an animal.
 15. A methodfor reducing symptoms associated with toxic shock comprising:administering a vaccine according to claim 10 to an animal.
 16. A mutantSPE-A toxin or fragment thereof, wherein the mutant has at least twoamino acid changes and is substantially nonlethal compared with aprotein substantially corresponding to wild type SPE-A toxin.
 17. Themutant SPE-A toxin of claim 16, wherein the mutant has at least one ofthe following characteristics: the mutant has a decrease in mitogenicityfor T-cells, the mutant does not substantially enhance endotoxin shock,the mutant is not lethal, or the mutant is nonlethal but retainsmitogenicity comparable to that of the wild type SPE-A toxin.
 18. Avaccine for protecting animals against at least one biological activityof wild-type SPE-A comprising: an effective amount of at least onemutant SPE-A toxin according to claim
 16. 19. A pharmaceuticalcomposition comprising: a mutant SPE-A according to claim 16 inadmixture with a physiologically acceptable carrier.
 20. A DNA sequenceencoding a mutant SPE-A toxin according to claim
 16. 21. A stablytransformed host cell comprising a DNA sequence according to claim 29.22. A method for protecting an animal against at least one biologicalactivity of a wild type SPE-A comprising: administering a vaccineaccording to claim 18 to an animal.
 23. A method for reducing symptomsassociated with toxic shock comprising: administering a vaccineaccording to claim 18 to an animal.